Methods for Treating and Detecting Sepsis in Humans

ABSTRACT

Biomarkers for identifying sepsis in humans are presented herein, as are related methods, uses, agents, and kits comprising same. Methods for treating, detecting, and diagnosing sepsis in humans are presented herein.

RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 16/526,923, filed Jul. 30, 2019, which claims priority of andbenefit to U.S. Provisional Application No. 62/711,970, filed Jul. 30,2018, the entireties of each of which is incorporated herein byreference for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 25, 2019, isnamed 176395-010601_SL.txt and is 185,649 bytes in size.

FIELD OF INVENTION

Polynucleotides relating to circulating nucleic acids (CNAs) indicativeof sepsis are described herein, as are probes, methods, and kits fordetecting and diagnosing sepsis in humans. Also encompassed herein areprobes, methods, and kits for detecting CNAs described herein insamples. In a particular embodiment, the CNAs are detected in samplesisolated from humans.

BACKGROUND

Sepsis, also known as systemic inflammatory response syndrome, is aleading cause of mortality in mammals and is characterized by symptomsthat include fever, elevated heart and breathing rate, and organfailure. Even under the best of care, sepsis can lead to septic shockwhich is frequently fatal. Sepsis is linked to immune responses toinfection and more particularly to an excessive inflammatory immuneresponse. Bacterial infections are the most common infection associatedwith sepsis, but fungal, viral, and parasitic infections can also leadto sepsis. Sepsis arises from a complicated interactive network of thecausative agent (e.g., a bacterial infection) and the host immune systemresponse to the agent. The genetic background of the host and the statusof the host immune system contribute to the potential for developingsepsis and severity of the condition once developed. Early phases ofsepsis are typically associated with high levels of inflammation due tothe release of inflammatory cytokines, such as high mobility group box 1protein (HMGB1) and tumor necrosis factor-alpha (TNF-α), interleukin-1(IL-1), and interleukin-6 (IL-6). The dramatic release of suchinflammatory cytokines, wherein a positive feedback loop is establishedbetween the cytokines and white blood cells, is referred to as acytokine storm (also known hypercytokinemia or cytokine cascade). Laterphases of sepsis are somewhat paradoxically associated with prolongedperiods of reduced immune system activity.

SUMMARY

Methods, reagents, and kits described herein relate to predicting and/ordiagnosing sepsis in a human in advance of the appearance of sepsissymptoms in the human. In accordance with the present experimentalfindings, methods, reagents, and kits described herein can be used topredict/diagnose sepsis in a human subject in advance of symptomaticpresentation as well as in later stages of the disease progress. Indeed,results presented herein demonstrate that methods, reagents, and kitsdescribed herein can diagnose sepsis in a human subject at least threedays in advance of clinical presentation. In light of results presentedin the examples, over-representation or under-representation of at leastone polynucleotide relative to an internal reference region in abiological sample or body fluid sample (e.g., serum), wherein the atleast one polynucleotide comprises any one of SEQ ID NOs: 1-57 or94-148, is a positive indicator that a human subject from which thesample was isolated is developing sepsis. Accordingly, the methods,reagents, and kits described herein provide for diagnosis of sepsis atearly, pre-symptomatic stages of the disease, as well as later stages ofthe disease. Methods for determining over-representation orunder-representation of at least one polynucleotide (CNA) relative to aninternal reference region, wherein the at least one polynucleotidecomprises any one of SEQ ID NOs: 1-57 or 94-148, in body fluids are alsodisclosed, as are kits for such purposes, methods for screening thediagnostic target, and detection tests for screening. In a particularembodiment, the internal reference regions is at least one of SEQ IDNOs: 59, 61, or 68.

In a particular aspect, a method is presented comprising administeringto a human identified as having sepsis a therapeutically effectiveamount of at least one agent used to treat sepsis, wherein the human isidentified as having sepsis by analyzing a biological sample isolatedfrom the human for over-representation or under-representation of atleast one polynucleotide relative to an internal reference region,wherein the at least one polynucleotide comprises any one of SEQ ID NOs:1-57 or 94-148 and wherein the over-representation orunder-representation of the at least one polynucleotide in thebiological sample is indicative of sepsis, thereby identifying the humanas having sepsis.

In another particular aspect, a method is presented comprisingadministering to a human identified as having over-representation orunder-representation of at least one polynucleotide relative to aninternal reference region a therapeutically effective amount of at leastone agent used to treat sepsis, wherein the human is identified ashaving over-representation or under-representation of at least onepolynucleotide relative to an internal reference region by analyzing abiological sample isolated from the human for over-representation orunder-representation of at least one polynucleotide relative to aninternal reference region, wherein the at least one polynucleotidecomprises any one of SEQ ID NOs: 1-57 or 94-148 and wherein theover-representation or under-representation of the at least onepolynucleotide in the biological sample is a positive indicator that thehuman is in need of the administering.

In another particular aspect, a method is presented for detecting sepsisin a human, comprising

(a) analyzing a biological sample isolated from the human forover-representation or under-representation of at least onepolynucleotide relative to an internal reference region, wherein the atleast one polynucleotide comprises any one of SEQ ID NOs: 1-57 or94-148, by contacting the biological sample with at least one syntheticprobe specific for a polynucleotide comprising any one of SEQ ID NOs:1-57 or 94-148, wherein the contacting generates complexes of syntheticprobes bound to specific polynucleotides when at least onepolynucleotide comprising any one of 1-57 or 94-148 is present in thebiological sample, (b) detecting the complexes of synthetic probes boundto specific polynucleotides, and detecting the internal reference regionin the biological sample, and (c) comparing the at least onepolynucleotide comprising any one of SEQ ID NOs: 1-57 or 94-148 detectedin the biological sample to the internal reference region detected inthe biological sample to determine relative over-representation andunder-representation of the at least one polynucleotide in thebiological sample, wherein detection of the over-representation andunder-representation of the at least one polynucleotide serves as apositive indicator of sepsis in the human.

In another particular aspect, a method is presented for detecting sepsisin a human, comprising analyzing a biological sample isolated from thehuman for over-representation or under-representation of at least onepolynucleotide comprising any one of SEQ ID NOs: 1-57 or 94-148 relativeto an internal reference region, wherein detection ofover-representation or under-representation of the at least one saidpolynucleotide relative to the internal reference region in thebiological sample is a positive indicator of sepsis in the human.

In another particular aspect, a method is presented for detecting sepsisin a human, comprising detecting at least one polynucleotide comprisingany one of SEQ ID NOs: 1-57 or 94-148 in a biological sample obtainedfrom the human wherein over-representation and under-representation ofthe at least one polynucleotide relative to an internal reference regionin the biological sample is a positive indicator of sepsis in the human.The method may further comprising use of a therapeutically effectiveamount of at least one agent used to treat sepsis for treatment of thehuman.

In another particular aspect, a method is presented for evaluatingrepresentation of at least one polynucleotide comprising any one of SEQID NOs: 1-57 or 94-148 in a biological sample of a human, the methodcomprising: analyzing the biological sample of the human forover-representation or under-representation of at least onepolynucleotide relative to an internal reference region, wherein the atleast one polynucleotide comprises any one of SEQ ID NOs: 1-57 or94-148, and wherein the over-representation or the under-representationof the at least one polynucleotide in the biological sample isdetermined by detecting the at least one polynucleotide comprising anyone of SEQ ID NOs: 1-57 or 94-148 in the biological sample, wherein thedetecting is achieved by contacting the biological sample with at leastone reagent that specifically binds to any one of SEQ ID NOs: 1-57 or94-148, detecting the internal reference region in the biologicalsample, and comparing the at least one polynucleotide comprising any oneof SEQ ID NOs: 1-57 or 94-148 detected in the biological sample to theinternal reference region detected in the biological sample to determinerelative over-representation and under-representation of the at leastone polynucleotide in the biological sample.

In a particular embodiment of any of the above methods, wherein the atleast one polynucleotide comprises any one of SEQ ID NOs: 1-57 or 94-148over-represented or under-represented relative to the internal referenceregion, the at least one is at least two, at least three, at least four,at least five, at least six, at least seven, at least eight, at leastnine, at least ten, at least eleven, at least twelve, at least thirteen,at least fourteen, at least fifteen, at least sixteen, at leastseventeen, at least eighteen, at least nineteen, at least twenty, atleast twenty-one, at least twenty-two, at least twenty-three, or atleast twenty-four of the polynucleotides comprising any one of SEQ IDNOs: 1-57 or 94-148.

In a particular embodiment of any of the above methods, the biologicalsample is blood, a product derived from blood, or a fraction derivedfrom blood. In a more particular embodiment, the product derived fromblood is plasma or serum.

In a particular embodiment of any of the above methods, the internalreference region comprises at least one polynucleotide comprising anyone of SEQ ID NOs: 59, 61, or 68.

In a particular embodiment of any of the above methods, detecting theover-representation or under-representation of the at least onepolynucleotide relative to an internal reference region comprises atleast one of a polymerase chain reaction (PCR)-based detection method, ahybridization-based method, enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), solid-phase enzyme immunoassay (EIA), massspectrometry, or microarray analysis. In a more particular embodiment,the PCR-based detection method is performed using at least one primerpair, wherein each primer pair of the at least one primer pair isspecific for any one of SEQ ID NOs: 1-57 or 94-148, and a primer pairspecific for at least one of SEQ ID NOs: 59, 61, or 68. In a moreparticular embodiment, the primer pair specific for any one of SEQ IDNOs: 1-57 or 94-148 and the primer pair specific for at least one of SEQID NOs: 59, 61, or 68 is any one of the primer pairs presented in Tables1-3. In another particular embodiment, the method further comprisessequencing amplification products corresponding to any one of SEQ IDNOs: 1-57 or 94-148 or at least one of SEQ ID NOs: 59, 61, or 68generated by the PCR-based detection method. In another particularembodiment, the SEQ ID NOs: (nucleic acid sequences) comprisecirculating nucleic acid. In another particular embodiment, the at leastone agent used to treat sepsis comprises at least one of an antibiotic,anti-fungal agent, anti-viral agent, anti-parasitic agent, or fluidssuitable for intravenous administration. In another particularembodiment, the human is monitored for sepsis. In another particularembodiment, the over-representation or under-representation of the atleast one polynucleotide relative to an internal reference region isdetermined using reagents comprising an antibody or a nucleic acid probespecific for any one of SEQ ID NOs: 1-57 or 94-148 and at least one ofSEQ ID NOs: 59, 61, or 68. In another particular embodiment, the nucleicacid probe specific for any one of SEQ ID NOs: 1-57 or 94-148 and atleast one of SEQ ID NOs: 59, 61, or 68 is labeled with a detectablelabel.

In another aspect, a probe comprising a manmade nucleotide sequence thatbinds specifically to a polynucleotide comprising any one of SEQ ID NOs:1-57 or 94-148 and at least one manmade tag conjugated thereto and atleast one of SEQ ID NOs: 59, 61, or 68 and at least one manmade tagconjugated thereto, wherein the manmade nucleotide sequence iscomplementary to the polynucleotide comprising any one of SEQ ID NOs:1-57 or 94-148 and at least one of SEQ ID NOs: 59, 61, or 68. In aparticular embodiment, the manmade nucleotide sequence that bindsspecifically to a polynucleotide comprising any one of SEQ ID NOs: 1-57or 94-148 and at least one of SEQ ID NOs: 59, 61, or 68 exhibits atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, or atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%complementarity to any one of SEQ ID NOs: 1-57 or 94-148 and at leastone of SEQ ID NOs: 59, 61, or 68. In a particular embodiment, themanmade tag is a detectable marker. In a more particular embodiment, thedetectable marker comprises a radioactive marker or fluorescent marker.In another embodiment, an array comprising at least one probe that bindsspecifically to any one of SEQ ID NOs: 1-57 or 94-148 and at least oneof SEQ ID NOs: 59, 61, or 68 is presented, wherein the at least oneprobe is bound to a solid surface. In a particular embodiment, the arrayis used for diagnosing sepsis. In another embodiment, a kit comprisingat least one probe that binds specifically to any one of SEQ ID NOs:1-57 or 94-148 and at least one of SEQ ID NOs: 59, 61, or 68 ispresented, wherein the kit further optionally comprises instructions foruse thereof. In a particular embodiment, the kit is used for diagnosingsepsis. In another particular embodiment, the kit is used for detectingthe indicated SEQ ID NOs:. In embodiments thereof, the array or kitcomprises comprising at least four, five, or ten different probescomprising a manmade nucleotide sequence that binds specifically to apolynucleotide comprising any one of the SEQ ID NOs: listed in Table 4and at least one manmade tag conjugated thereto, wherein the manmadenucleotide sequence is complementary to the polynucleotide comprisingany one of the SEQ ID NOs: listed in Table 4. In a particularembodiment, the array comprises a microarray, gene chip, DNA chip, or aFILMARRAY®.

In another aspect, a primer comprising, consisting essentially of, orconsisting of a manmade nucleotide sequence that binds specifically to apolynucleotide comprising any one of the SEQ ID NOs: listed in Table 4and at least one manmade tag conjugated thereto is presented, whereinthe manmade nucleotide sequence is any one of the polynucleotide primersequences or a primer pair listed in Tables 1-3 or a variant thereof. Ina more particular embodiment, the variant of any one of thepolynucleotide primer sequences listed in Tables 1-3 is at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, or at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to anyone of the polynucleotide sequences listed in Table 4. In anotherparticular embodiment, the manmade tag is a detectable marker (e.g., aradioactive marker or fluorescent marker.

In another aspect, a kit for detecting sepsis in a human is presentedcomprising at least one primer pair for amplifying a polynucleotidecomprising any one of the SEQ ID NOs: listed in Table 4, wherein the atleast one primer pair is listed in Tables 1-3 and optionally,instructions for use thereof. In a particular embodiment, the at leastone primer pair is four primer pairs and wherein each primer pair of thefour primer pairs specifically amplifies a different polynucleotidecomprising any one of the SEQ ID NOs: listed in Table 4. In anotherparticular embodiment, the at least one primer pair is five primer pairsand wherein each primer pair of the five primer pairs specificallyamplifies a different polynucleotide comprising any one of the SEQ IDNOs: listed in Table 4. In another particular embodiment, the at leastone primer pair is ten primer pairs and wherein each primer pair of theten primer pairs specifically amplifies a different polynucleotidecomprising any one of the SEQ ID NOs: listed in Table 4.

In another aspect, use of a polynucleotide sequence comprising any oneof the SEQ ID NOs: listed in Table 4 as a biomarker for the detection ofsepsis in a human is encompassed herein. In a particular embodiment, thebiomarker is a biomarker in a sample obtained from the human, whereinthe sample is a sample of blood, a product derived from blood or afraction derived from blood (e.g. plasma or serum)

In an aspect, a method for treating a human suspected of having sepsisis presented, the method comprising treating the human identified ashaving sepsis with a therapeutically effective amount of at least oneagent used to treat sepsis, wherein the human is identifiable as havingsepsis by analyzing a biological sample isolated from the human forover-representation or under-representation of at least onepolynucleotide, wherein the at least one polynucleotide comprises anyone of SEQ ID NOs: 1-57 or 94-148 and wherein the over-representation orunder-representation of the at least one polynucleotide in thebiological sample is a positive indicator of sepsis. In a particularembodiment thereof, the over-representation or the under-representationof the at least one polynucleotide is determined relative to an internalreference region. In a particular embodiment, the internal referenceregions is at least one of SEQ ID NOs: 59, 61, or 68.

Also encompassed herein is a therapeutically effective amount of atleast one agent used to treat sepsis or a composition thereof for use ina method of treating sepsis in a human identified as having sepsis,wherein the human is identified as having sepsis by analyzing abiological sample isolated from the human for over-representation orunder-representation of at least one polynucleotide, wherein the atleast one polynucleotide comprises any one of SEQ ID NOs: 1-57 or 94-148and wherein the over-representation or under-representation of the atleast one polynucleotide in the biological sample is a positiveindicator of sepsis. The human may exhibit symptoms of sepsis or may beasymptomatic. In a particular embodiment thereof, theover-representation or the under-representation of the at least onepolynucleotide is determined relative to an internal reference region.In a particular embodiment, the internal reference regions is at leastone of SEQ ID NOs: 59, 61, or 68.

In another aspect, a method for treating a human identified asexhibiting over-representation or under-representation of at least onepolynucleotide is presented, wherein the at least one polynucleotidecomprises any one of SEQ ID NOs: 1-57 or 94-148 and wherein theover-representation or the under-representation of the at least onepolynucleotide in the biological sample is a positive indicator ofsepsis, the method comprising treating the human identified asexhibiting the over-representation or the under-representation of the atleast one polynucleotide with a therapeutically effective amount of atleast one agent used to treat sepsis. In a particular embodimentthereof, the over-representation or the under-representation of the atleast one polynucleotide is determined relative to an internal referenceregion. In another particular embodiment, the human is identifiable ashaving sepsis by analysis of a biological sample isolated from the humanfor the over-representation or the under-representation of the at leastone polynucleotide, wherein the over-representation or theunder-representation of the at least one polynucleotide may optionallybe determined relative to an internal reference region. In a particularembodiment, the internal reference regions is at least one of SEQ IDNOs: 59, 61, or 68.

In yet another aspect, a method for treating a human with early stagesepsis (e.g., pre-symptomatic sepsis) is presented, the methodcomprising treating the human with early stage sepsis with atherapeutically effective amount of at least one agent used to treatsepsis, wherein the human is identifiable as having early stage sepsisby analyzing a biological sample isolated from the human forover-representation or under-representation of at least onepolynucleotide, wherein the at least one polynucleotide comprises anyone of SEQ ID NOs: 1-57 or 94-148 and wherein the over-representation orunder-representation of the at least one polynucleotide in thebiological sample is a positive indicator of sepsis. In a particularembodiment thereof, the over-representation or the under-representationof the at least one polynucleotide is determined relative to an internalreference region. In a particular embodiment, the internal referenceregions is at least one of SEQ ID NOs: 59, 61, or 68.

Also encompassed herein is a therapeutically effective amount of atleast one agent used to treat sepsis or a composition thereof for use ina method of treating sepsis in a human identified with early stagesepsis, wherein the human is identified as having early stage sepsis byanalyzing a biological sample isolated from the human forover-representation or under-representation of at least onepolynucleotide, wherein the at least one polynucleotide comprises anyone of SEQ ID NOs: 1-57 or 94-148 and wherein the over-representation orunder-representation of the at least one polynucleotide in thebiological sample is a positive indicator of sepsis. The human mayexhibit symptoms of sepsis or may be asymptomatic. In a particularembodiment thereof, the over-representation or the under-representationof the at least one polynucleotide is determined relative to an internalreference region.

Methods described herein comprise embodiments wherein the at least onepolynucleotide comprising any one of SEQ ID NOs: 1-57 or 94-148over-represented or under-represented is at least two, at least three,at least four, at least five, at least six, at least seven, at leasteight, at least nine, at least ten, at least eleven, at least twelve, atleast thirteen, at least fourteen, at least fifteen, at least sixteen,at least seventeen, at least eighteen, at least nineteen, or at leasttwenty of the polynucleotides comprising SEQ ID NOs: 1-57 or 94-148. Ina particular embodiment thereof, the over-representation or theunder-representation of the at least two, at least three, at least four,at least five, at least six, at least seven, at least eight, at leastnine, at least ten, at least eleven, at least twelve, at least thirteen,at least fourteen, at least fifteen, at least sixteen, at leastseventeen, at least eighteen, at least nineteen, or at least twenty ofthe polynucleotides comprising SEQ ID NOs: 1-57 or 94-148 is determinedrelative to an internal reference region. In a particular embodiment,the internal reference regions is at least one of SEQ ID NOs: 59, 61, or68.

In a further aspect, a method for evaluating representation of at leastone polynucleotide comprising any one of SEQ ID NOs: 1-57 or 94-148 in abiological sample of a human is presented, the method comprising:

analyzing the biological sample of the human for over-representation orunder-representation of at least one polynucleotide, wherein the atleast one polynucleotide comprises any one of SEQ ID NOs: 1-57 or94-148, and

wherein the over-representation or the under-representation of the atleast one polynucleotide in the biological sample is determined bydetecting the at least one polynucleotide comprising any one of SEQ IDNOs: 1-57 or 94-148 in the biological sample, wherein the detecting isachieved by contacting the biological sample with at least one reagentthat specifically binds to any one of SEQ ID NOs: 1-57 or 94-148, todetermine relative over-representation and under-representation of theat least one polynucleotide in the biological sample. In a particularembodiment thereof, the over-representation or the under-representationof the at least one polynucleotide in the biological sample, whencompared to an internal reference is determined; for example byquantitative real time polymerase chain reaction (RT-PCR). In accordancewith methods described herein, amplification of regions of the CNAsrepresented by SEQ ID NOs: 1-57 or 94-148 involves a determination ofhow many amplification cycles are called for to reach a desireddetection limit. In another particular embodiment, theover-representation or the under-representation of the at least onepolynucleotide in the biological sample is determined by detecting theat least one polynucleotide comprising any one of SEQ ID NOs: 1-57 or94-148 in the biological sample, wherein the detecting is achieved bycontacting the biological sample with at least one reagent thatspecifically binds to any one of SEQ ID NOs: 1-57 or 94-148, anddetecting the internal reference region in the biological sample, andcomparing the at least one polynucleotide comprising any one of SEQ IDNOs: 1-57 or 94-148 detected in the biological sample to the internalreference region detected in the biological sample to determine relativeover-representation and under-representation of the at least onepolynucleotide in the biological sample. The over-representation orunder-representation of the at least one polynucleotide may also bereferred to herein as a deviation of the level of the at least onepolynucleotide in the biological sample of an infected subject relativeto the level of the at least one polynucleotide in a biological sampleof a healthy subject.

Detection of the at least one polynucleotide may be achieved usingRT-PCR as detailed herein as well as amplification free detectionmethods.

Methods described herein comprise embodiments wherein the biologicalsample is a bodily fluid, such as, without limitation, whole blood, ablood fraction, saliva, urine, sputum, cerebrospinal fluid, tears,sweat, milk, or interstitial fluid. In a particular embodiment, thebiological sample is blood, a product derived from blood, or a fractionderived from blood. In a particular embodiment, the product derived fromblood is plasma or serum.

Methods described herein comprise embodiments wherein detecting theover-representation or under-representation of the at least onepolynucleotide comprises at least one of a PCR-based detection method(e.g., RT-PCR), a hybridization-based method, enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (MA), solid-phase enzymeimmunoassay (EIA), mass spectrometry, and microarray analysis. In aparticular embodiment thereof, detecting the over-representation orunder-representation of the at least one polynucleotide is determinedrelative to an internal reference region. In a particular embodiment,the internal reference regions is at least one of SEQ ID NOs: 59, 61, or68.

In embodiments comprising a PCR-based detection method, the PCR-baseddetection method comprises amplifying nucleic acid sequences in thebiological sample using primers that are specific for and capable ofamplifying any one of SEQ ID NOs: 1-57 or 94-148, wherein the amplifyinggenerates amplification products corresponding to any one of SEQ ID NOs:1-57 or 94-148 when the biological sample comprises any one of SEQ IDNOs: 1-57 or 94-148. In embodiments comprising a PCR-based detectionmethod, the PCR-based detection method is performed using at least oneprimer pair, wherein each primer pair of the at least one primer pair isspecific for any one of SEQ ID NOs: 1-57 or 94-148. In a more particularembodiment thereof, the primer pair specific for any one of SEQ ID NOs:1-57 or 94-148 is any one of the primer pairs presented in Table 1 orTable 3. In an even more particular embodiment thereof, the PCR-baseddetection method further comprises sequencing the amplification productscorresponding to any one of SEQ ID NOs: 1-57 or 94-148. In a particularembodiment, the internal reference regions is at least one of SEQ IDNOs: 59, 61, or 68 and primer pairs therefor are presented in Table 2.

Methods described herein comprise embodiments wherein nucleic acidsequences comprise circulating nucleic acids.

Methods described herein comprise embodiments wherein the at least oneagent used to treat sepsis comprises at least one of an antibiotic,anti-fungal agent, anti-viral agent, anti-parasitic agent, or fluidssuitable for intravenous administration.

Methods described herein comprise embodiments wherein the human ismonitored for sepsis.

Methods described herein comprise embodiments wherein theover-representation or under-representation of the at least onepolynucleotide is determined using reagents comprising an antibody or anucleic acid probe specific for any one of SEQ ID NOs: 1-57 or 94-148.In a particular embodiment thereof, the over-representation orunder-representation of the at least one polynucleotide is determinedrelative to an internal reference region. In a more particularembodiment, the antibody is a monoclonal or a polyclonal antibody. In astill more particular embodiment, the antibody is obtained from mice,rats, rabbits, goats, chicken, donkey, horses or guinea pigs.

Methods described herein comprise embodiments wherein a nucleic acidprobe specific for any one of SEQ ID NOs: 1-57 or 94-148 is labeled witha detectable label.

In another aspect, a probe comprising a manmade nucleotide sequencecapable of binding specifically to a polynucleotide comprising any oneof SEQ ID NOs: 1-57 or 94-148 and at least one manmade tag conjugatedthereto is presented, wherein the manmade nucleotide sequence iscomplementary to the polynucleotide comprising any one of SEQ ID NOs:1-57 or 94-148. In a particular embodiment thereof, the manmadenucleotide sequence capable of binding specifically to a polynucleotidecomprising any one of SEQ ID NOs: 1-57 or 94-148 exhibits at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%,at least 96%, at least 97%, at least 98%, or at least 99%complementarity to any one of SEQ ID NOs: 1-57 or 94-148. In anotherparticular embodiment, the manmade tag is a detectable marker. In astill more particular embodiment, the detectable marker comprises aradioactive marker or fluorescent marker.

Also encompassed herein is an array comprising at least one probedescribed herein, wherein the at least one probe is bound to a solidsurface. Such arrays may comprise a microarray, gene chip, DNA chip, aFILMARRAY®, or a similar array.

Kits comprising at least one probe described herein and instructions foruse thereof are also encompassed.

In a particular embodiment, the array or kit described herein comprisesat least two, at least three, at least four, at least five, at leastsix, at least seven, at least eight, at least nine, at least ten, atleast eleven, at least twelve, at least thirteen, at least fourteen, atleast fifteen, at least sixteen, at least seventeen, at least eighteen,at least nineteen, or at least twenty different probes comprising amanmade nucleotide sequence capable of binding specifically tocorresponding polynucleotides comprising any one of SEQ ID NOs: 1-57 or94-148 and at least one manmade tag conjugated thereto, wherein themanmade nucleotide sequence is complementary to the polynucleotidecomprising any one of SEQ ID NOs: 1-57 or 94-148. In a particularembodiment, the array or kit described herein comprises at least fourdifferent probes comprising a manmade nucleotide sequence capable ofbinding specifically to a polynucleotide comprising any one of SEQ IDNOs: 1-57 or 94-148 and at least one manmade tag conjugated thereto,wherein the manmade nucleotide sequence is complementary to thepolynucleotide comprising any one of SEQ ID NOs: 1-57 or 94-148. In amore particular embodiment, the array or kit described herein comprisesat least eight different probes comprising a manmade nucleotide sequencecapable of binding specifically to a polynucleotide comprising any oneof SEQ ID NOs: 1-57 or 94-148 and at least one manmade tag conjugatedthereto, wherein the manmade nucleotide sequence is complementary to thepolynucleotide comprising any one of SEQ ID NOs: 1-57 or 94-148.

Also encompassed herein is a primer comprising a manmade nucleotidesequence capable of binding specifically to a polynucleotide comprisingany one of SEQ ID NOs: 1-57 or 94-148 and at least one manmade tagconjugated thereto, wherein the manmade nucleotide sequence is any oneof the polynucleotide sequences listed in Table 1 or Table 3 as aforward primer or a variant thereof or as a reverse primer or a variantthereof. In an embodiment thereof, the variant of any one of the primersequences listed in Table 1 or Table 3 is at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, or at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identical to any one of theprimer sequences listed in Table 1 or Table 3. In a particularembodiment, a variant of a primer comprises different nucleotides at the5′ end of the primer, which positions are more tolerant of variationsthereto. In a particular embodiment, the manmade tag is a detectablemarker (e.g., a radioactive marker, fluorescent dye), a tag that isspecifically recognized (e.g., bound) by a labeled reagent (e.g., alabeled antibody), a tag that is specifically bound by a magnetic bead,or any other marker comprising detectable label.

Also encompassed herein are primers and probes for use in detecting theat least one polynucleotide described herein, wherein when using aTaqMan system, the detection of a particular polynucleotide sequencerequires two specific primers (a forward and reverse primer pairspecific for the polynucleotide sequence) and a probe (labeledoligonucleotide specific for the polynucleotide sequence). A labeledprobe may, for example, be a TaqMan probe comprising a fluorescent labelat a first terminus and a quencher at a second terminus whereby uponprobe displacement during quantitative PCR (qPCR), the probe is cleaved,thus releasing the fluorescent label from the vicinity and effects ofthe quencher. Upon release from the vicinity/effects of the quencher, ahigher fluorescence is emitted from the fluorescent label, which can bedetected and quantitated to reflect the level/amount of the particularpolynucleotide sequence present in a sample.

Also encompassed herein are methods for amplification-free detection ofthe at least one polynucleotide described herein, wherein such methodscomprise a pair of oligonucleotide probes which are specific for theparticular polynucleotide and are differentially labeled such that wheneach is bound to the polynucleotide and therefore in close proximity,the signal emitted from the probes changes. With respect tooligonucleotide probes that are labeled with fluorescent tags, closeproximity of the fluorescent tags when bound to the polynucleotide isdetected by an increase in fluorescence emissions.

Also encompassed herein is a primer consisting essentially of orconsisting of a manmade nucleotide sequence capable of bindingspecifically to a polynucleotide comprising any one of SEQ ID NOs: 1-57or 94-148 and at least one manmade tag conjugated thereto, wherein themanmade nucleotide sequence is any one of the polynucleotide sequenceslisted in Table 1 or Table 3 or a variant thereof. In an embodimentthereof, the variant of any one of the polynucleotide sequences listedin Table 1 or Table 3 is at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, or at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to any one of the polynucleotidesequences listed in Table 1 or Table 3. In a particular embodiment, themanmade tag is a detectable marker (e.g., a radioactive marker orfluorescent marker).

Primer pairs complementary to SEQ ID NOs: 1-57 or 94-148 and suitablefor PCR amplification are readily determined based on the sequences ofSEQ ID NOs: 1-57 or 94-148 (5′ to 3′ strands) and reverse strandsthereof (3′ to 5′ strands). Such primers are typically 8-20 nucleotidesin length and are complementary (e.g., exhibit perfect complementarityor may be variants thereof that maintain a degree of complementaritysufficient to bind and act as primers in a PCR amplification) to any oneof SEQ ID NOs: 1-57 or 94-148 or a reverse strand thereof. Choicesregarding primer pairs suitable for PCR amplification are alsodetermined based on the guanine/cytosine content of a potential primersequence and the distance between the primers in a pair when bound tothe target polynucleotide, with the understanding that PCR amplificationproducts must be of a detectable size.

In another embodiment, therapeutic efficacy of a treatment regimen maybe evaluated based on a change in the over-representation orunder-representation of at least one of SEQ ID NOs: 1-57 or 94-148following onset of the treatment regimen. In a particular embodiment, adecrease in representation of a CNA that is over-represented in sepsis(e.g., SEQ ID NOs: 94-148) is indicative that the treatment regimen istherapeutically effective. In another particular embodiment, an increasein representation of a CNA that is under-represented in sepsis (e.g.,SEQ ID NOs: 1-57) is indicative that the treatment regimen istherapeutically effective.

Also encompassed herein is an array comprising at least one primerlisted in Table 1 or Table 3, wherein the at least one primer is boundto a solid surface.

A kit for detecting sepsis in a human is also described, wherein the kitcomprises at least one primer pair for amplifying a polynucleotidecomprising any one of SEQ ID NOs: 1-57 or 94-148, wherein the at leastone primer pair is listed in Table 1 or Table 3 or a functional variantthereof or a primer that may be determined based on any one of thesequences designated herein as SEQ ID NOs: 1-57 or 94-148 andinstructions for use thereof. In a particular embodiment, the at leastone primer pair is four primer pairs and each primer pair of the fourprimer pairs specifically amplifies a different polynucleotidecomprising any one of SEQ ID NOs: 1-57 or 94-148. In another particularembodiment, the at least one primer pair is eight primer pairs and eachprimer pair of the eight primer pairs specifically amplifies a differentpolynucleotide comprising any one of SEQ ID NOs: 1-57 or 94-148.

Additional aspects of the present invention will be apparent in view ofthe description which follows.

Additional aspects of the present invention will be apparent in view ofthe description which follows.

BRIEF DESCRIPTION OF THE FIGURES AND TABLES

The invention will now be described in relation to the drawings andtables.

FIGS. 1A-H presents Table 1, wherein each of the sequences correspondsto a CNA and is designated SEQ ID NO: 1-57 in consecutive order. SEQ IDNOs: 1-57 are positive indicators of sepsis that are more highlyrepresented in healthy controls and thus, a decrease in copy number isobserved during infection. Accordingly, a decrease in copy number of anyone of SEQ ID NOs: 1-57 is a positive indicator of sepsis. Forward andreverse primers suitable for PCR amplification of each of SEQ ID NOs:1-57 are presented in the same row as the CNA for which they arespecific.

FIGS. 2A-F presents Table 2, wherein each of the Sequences correspondsto a CNA and is designated SEQ ID NO: 58-93 in consecutive order. SEQ IDNOs: 58-93 are equally represented in copy number in healthy controlsand subjects afflicted with sepsis. Forward and reverse primers suitablefor PCR amplification of each of SEQ ID NOs: 58-93 are presented in thesame row as the CNA for which they are specific.

FIGS. 3A-I presents Table 3, wherein each of the Sequences correspondsto a CNA and is designated SEQ ID NO: 94-148 in consecutive order. SEQID NOs: 94-148 are positive indicators of sepsis that increase in copynumber during infection. Forward and reverse primers suitable for PCRamplification of each of SEQ ID NOs: 94-148 are presented in the samerow as the CNA for which they are specific.

DETAILED DESCRIPTION OF THE INVENTION

Various embodiments will be described in detail with reference to thedrawings and tables. Reference to various embodiments does not limit thescope of the claims attached hereto. Additionally, any examples setforth in this specification are not intended to be limiting and merelyset forth some of the many possible embodiments.

Methods, reagents, and kits described herein relate to detection andanalysis of cell-free DNA available in the blood following activesecretion, cell death or apoptosis. The present inventors havedemonstrated that cell-free DNA indicative of sepsis can be detected inblood up to 2-3 days prior to clinical diagnosis of sepsis according toSepsis-3-definition (defined below in accordance with current medicalstandards). Traditional biomarkers such as lactate, procalcitonin andothers are measured only after clinical sepsis is evident from thepatient's vital signs or labs. In high-risk patients, this may be toolate to prevent the body's cascade of cytokine release, the harbinger ofdevelopment for sepsis sequelae.

Accordingly, the lead-time provided by the present methods, reagents,and kits identifies subjects requiring heightened attention andmonitoring. In particular embodiments, the present methods, reagents,and kits provide information on which basis a medical practitioner mayinitiate appropriate therapy and identify the site of infection, whichmay require source control, before the onset of irreversible organdysfunction and widespread cellular hypoxia that occurs during the laterstages of sepsis in septic patients.

Further to the above, methods described herein are based on the analysisof circulating nucleic acids, which are present in, for example, serumand plasma of humans. The composition and relative abundance ofcirculating nucleic acids varies depending on disease state. The presentinventors identified particular genomic regions that do not change inrelative abundance of specific regions of circulating nucleic acidsbetween sepsis patients and controls (non-sepsis), which genomic regionsare designated internal reference regions. Genomic regions thatsignificantly vary in abundance between sepsis patients and controlswere also identified and designated disease motifs. Relative abundancefor disease motifs can be examined using Real-Time Polymerase ChainReaction (RT-PCR) to determine if an “unknown” sample can be identifiedas exhibiting features characteristic of sepsis or featurescharacteristic of non-sepsis. In a particular embodiment, identifying asample as exhibiting features characteristic of sepsis may be used topredict onset of sepsis or diagnose sepsis.

It is to be understood that combinations of primers binding to genomicregions that significantly vary in coverage and abundance between sepsispatients can be optimized. The RT-PCR assay can be designed based atleast in part on primers used and such an assay can be used to identifyearly features of sepsis.

Probes, methods, and kits described herein can analyze cell free DNA(cfDNA) collected at a preclinical stage of sepsis and accuratelydetermine which patients will go on to develop sepsis. This preclinicalphase includes patients that already have molecular evidence of aninfection but have not yet manifested traditional criteria includingvital sign abnormalities, fever and laboratory changes (according to theSepsis-3-definition).

Early identification of sepsis, formerly known as septicaemia, isclinically valuable because sepsis has a high morbidity and mortalityrate in mammals in general and, even under the best of care, in humans.Sepsis is characterized as a systemic, excessive inflammatory response,which is frequently triggered by infection and proceeds unchecked bynormal regulatory controls. Sepsis is most often associated withbacterial infections in humans. Severe trauma can also lead to systemicresponses that adversely affect adaptive and innate immune responses,leading to excessive inflammation having pathological consequences.Severe trauma patients, such as burn and accident victims, maysubsequently be afflicted with infections, some of which lead to sepsis.Clinical symptoms of sepsis in humans include lethargy,fever/hyperthermia in early stages of sepsis, confusion, and/orhypothermia in end-stage sepsis (e.g., septic shock).

Sepsis is a leading cause of hospitalization in the United States and isone of the most expensive conditions to treat because it typicallyrequires a stay in the Intensive Care Unit (ICU) to achieve patientrecovery. It has been estimated that 19 million patients will developsepsis worldwide each year and this estimate likely substantiallyunderestimates the problem. Indeed, sepsis is a leading cause of death,morbidity, and medical expense. Sepsis contributes to one-third ofdeaths of hospitalized patients. The mortality rate is estimated to bebetween 20% and 80%, depending on the cause of the sepsis andprogression of sepsis. Onset of sepsis in patients following a hospitalstay is frequent and particularly challenging, at least in part, becausesuch patients are likely still in a state of recovery and therefore,physiologically weakened. Such patients may be in early, pre-clinicalstages of sepsis upon release, but absent the present discoveries,sepsis cannot be detected at pre-clinical stages of the disease.Re-admittance to hospitals is a significant problem as it is costly andtypically not covered in full by medical insurance policies. Indeed,people with sepsis are two to three times more likely to be readmittedto the hospital as people released from the hospital who were diagnosedwith a variety of conditions, including heart failure, pneumonia, andchronic obstructive pulmonary disease. It is also noteworthy that a boutof sepsis can have lifelong ramifications for patients who aresuccessfully treated and survive. As the number of sepsis survivorsgrows, it has become increasingly apparent that sepsis frequently leadsto long-lasting physical and cognitive impairment.

Indeed, the growing number of patients who survive the acute phase ofsepsis has revealed that the danger associated with the disease extendslong after discharge from the hospital. Long-term mortality following about of sepsis is approximately 50% in the first year and rises to >81%over five years post-discharge. Sepsis survivors are also more prone toexhibit diminished physical and/or cognitive function following the boutof sepsis than age-matched controls hospitalized for unrelatedindications. As a consequence of these diminished functions, many sepsissurvivors are unable to return to the workforce.

The standard of care for treatment of bacterial sepsis in humans callsfor aggressive use of antimicrobial therapy (e.g., antibiotics,anti-fungal agent, anti-viral agent), anti-parasitic agent therapy,and/or intravenous fluid therapy, which may be administered inconjunction with anti-inflammatory therapy. The choice of antibioticused for treating a septic patient depends on the type of bacteria thatinfected the subject, which infection subsequently led to sepsis in thesubject. Methods for culturing and identifying bacteria in samplesisolated from a subject are known in the art. Broad-spectrum antibioticsare recommended within one hour after sepsis recognition/diagnosis.Beta-lactam antibiotics having broad coverage administered incombination with fluoroquinolones, macrolides, or aminoglycosides arerecommended for severe cases of sepsis. An exemplary inflammatorytherapy involves administration of non-steroidal anti-inflammatoryagents to a human afflicted with bacterial sepsis.

Fungal infections are treated with anti-fungal medications specific tothe particular fungus that caused the infection. These may be used in acream or ointment, suppository, or pill form. Candida species are thepredominant agent of fungal sepsis and account for about 5% of all casesof severe sepsis and septic shock in the United States. The antifungalarmamentarium has expanded considerably with the advent of lipidformulations of amphotericin B, the newest triazoles and theechinocandins. Clinical trials have shown that the triazoles andechinocandins are efficacious and well tolerated antifungal therapies.

Prospective treatments for specific viruses implicated in sepsis arebeing developed. Pleconaril is an antiviral against enteroviralinfection which inhibits viral attachment to the hosts cell receptorsand prevents uncoating of the viral nucleic acids. Examples of ativiralagents that may be beneficial in presentations of sepsis in certainsituations include acyclovir, which has been proven effective in HSVinfections, amantadine, rimantadine, oseltamivir, and zanamivir forinfluenza, and more broad-spectrum antiviral drugs like ribavirin andfavipiravir.

Parasite infections can lead to sepsis. For Giardia infections, amedication like metronidazole (Flagyl), tinidazole (Tindamax) ornitazoxanide (Alinia) may be used. For Chagas disease, antiparasiticmedications such as benznidazole and nifurtimox may be used. Fortapeworm, the most commonly used medications are praziquantel(Biltricide), aibendazole (Albenza), and nitazoxanide (Alinia). However,if the infection has progressed and become more invasive, you may needtreatment with anti-inflammatory medications, anti-seizure medications,a shunt to drain fluid from your brain, or surgery to remove cystscaused by the tapeworm. For roundworm, the most commonly usedmedications include medendazole (Vermox), albdendazole (Albenza) andivermectin (Stromectol). Surgery could be required to remove the worm ifthere is a bowel obstruction.

The following guidelines provide the standard of care for sepsispatients as established by the American Medical Association:

Managing Infection:

Antibiotics: Administer broad-spectrum intravenous antimicrobials forall likely pathogens within 1 hour after sepsis recognition (strongrecommendation; moderate quality of evidence [QOE]).

Source control: Obtain anatomic source control as rapidly as ispractical (best practice statement [BPS]).

Antibiotic stewardship: Assess patients daily for de-escalation ofantimicrobials; narrow therapy based on cultures and/or clinicalimprovement (BPS).

Managing Resuscitation:

Fluids: For patients with sepsis-induced hypoperfusion, provide 30 mL/kgof intravenous crystalloid within 3 hours (strong recommendation; lowQOE) with additional fluid based on frequent reassessment (BPS),preferentially using dynamic variables to assess fluid responsiveness(weak recommendation; low QOE).

Resuscitation targets: For patients with septic shock requiringvasopressors, target a mean arterial pressure (MAP) of 65 mm Hg (strongrecommendation; moderate QOE).

Vasopressors: Use norepinephrine as a first-choice vasopressor (strongrecommendation; moderate QOE).

Mechanical Ventilation in Patients with Sepsis-Related Acute RespiratoryDistress Syndrome (ARDS):

Target a tidal volume of 6 mL/kg of predicted body weight (strongrecommendation; high QOE) and a plateau pressure of ≤30 cm H₂O (strongrecommendation; moderate QOE). See, for example, Howell and Davis (2017,JAMA 317:847).

As indicated hereinabove, sepsis is associated with multiple organfailure and high mortality. Commonly used markers for diagnosis ofsepsis include: elevated leukocyte counts and elevated cytokine levelssuch as IL-6, IL-8 and IL-18, C-reactive protein, and procalcitonin. Thelatter two proteins are expressed at higher levels after trauma. Withrespect to procalcitonin, determination of procalcitonin levels isviewed as a helpful diagnostic marker, but is not viewed as a definitivemarker of sepsis. Soluble urokinase-type plasminogen activator receptor(SuPAR) is considered to be a nonspecific marker of inflammation, buthas prognostic value in the context of sepsis because higher SuPARlevels are associated with higher mortality in sepsis patients.

None of these markers provides a predictive indicator of sepsis, butrather collectively serve as indicators only upon onset of sepsis.Indeed, in advance of the present findings, a reliable method to predictrisk for or diagnose sepsis in advance of the onset of clinical symptomsdid not exist. As detailed herein, the methods, reagents, and kitsdescribed herein make it possible to diagnose patients with sepsis atpre-clinical stages and thus, make it possible to treat such patients inadvance of the onset of clinical symptoms and at the least reduce theseverity of acute sepsis in the patient should the patient progresstoward disease despite early intervention.

Exemplary Target Patient Populations

Adult patients admitted to, for example, a hospital who are at high-riskfor developing sepsis include the following:

-   -   Victims of trauma with either an Injury Severity Score of ≥15 or        a Glasgow Coma Score of ≤8 on hospital presentation;    -   Any patient undergoing high-risk surgical procedures including        any emergency surgery, high risk elective surgery procedures        involving the thorax, esophagus, stomach, small bowel, large        bowel; or    -   Any patient being admitted to any intensive care unit (ICU)        setting for any reason with no current evidence or suspicion of        active infection as determined by primary team of attending        medical practitioners.

Methods, reagents, and kits described herein relate to predicting and/ordiagnosing sepsis in a human in advance of the appearance of symptoms ofsepsis in the human. In accordance with the experimental findingspresented herein, methods, reagents, and kits described are useful fordiagnosing sepsis in a human subject in advance of symptomaticpresentation. Indeed, results presented herein demonstrate that methods,reagents, and kits described herein can diagnose sepsis in a humansubject at least three days in advance of clinical presentation. Inlight of results presented in the Examples, detection ofover-representation or under-representation of at least onepolynucleotide relative to an internal reference region in a body fluidsample (e.g., serum) isolated from a human subject, wherein the at leastone polynucleotide comprises any one of SEQ ID NOs: 1-57 or 94-148, is apositive indicator that the human subject will develop sepsis symptoms.Accordingly, the methods, reagents, and kits described herein providefor diagnosis of sepsis at pre-clinical stages of the disease. Alsoencompassed herein are methods to assess over-representation orunder-representation of at least one polynucleotide relative to aninternal reference region, wherein the at least one polynucleotidecomprises any one of SEQ ID NOs: 1-57 or 94-148, in body fluids and kitsfor such purposes.

In addition to plasma or serum, over-representation orunder-representation of at least one polynucleotide (at least one of SEQID NOs: 1-57 or 94-148) relative to an internal reference region may bedetermined in other body fluids isolated from a human subject including:whole blood, a product derived from blood, or any fraction derived fromblood (in addition to plasma or serum.

Any known method may be used for the determination ofover-representation or under-representation of at least onepolynucleotide (at least one of SEQ ID NOs: 1-57 or 94-148) relative toan internal reference region in body fluids. Methods encompassed forsuch determinations include: polymerase chain reaction (PCR)amplification with sequence specific primer pairs, a hybridization-basedmethod, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay(RIA), solid-phase enzyme immunoassay (EIA), mass spectrometry,microarray analysis, and any combination thereof. Such methods when usedfor determining risk for developing sepsis or predicting onset of sepsisare encompassed herein.

In a particular method for determining over-representation orunder-representation of any one of SEQ ID NOs: 1-57 or 94-148 relativeto an internal reference region in human body fluids, e.g. serum, themethod calls for PCR amplification with sequence specific primer pairs.In an embodiment thereof, the PCR amplification is performed with atleast one primer pair specific for any one of SEQ ID NOs: 1-57 or94-148. Exemplary primer pairs for amplifying any one of SEQ ID NOs:1-57 or 94-148 are presented in Table 1 or Table 3. Conditions forperforming PCR amplifications are known in the art and presented in theExamples herein below. Such conditions may be determined based in parton the composition of a primer and/or primer pair and considerationsrelating to same are known in the art.

In another particular method for determining over-representation orunder-representation of any one of SEQ ID NOs: 1-57 or 94-148 relativeto an internal reference region in human body fluids, e.g. serum, themethod calls for an ELISA. In one embodiment, the ELISA for at least oneof SEQ ID NOs: 1-57 or 94-148 involves a sandwich array. In such anembodiment, PCR amplification of at least one of SEQ ID NOs: 1-57 or94-148 may be performed as an initial step. Conventional microtiterplates may be coated with a first antibody, e.g. a guinea pig polyclonalantibody, directed against any one of SEQ ID NOs: 1-57 or 94-148. Theplates are then blocked and the sample or reference is loaded. Afterincubation with, e.g., at least one of SEQ ID NOs: 1-57 or 94-148, asecond antibody against any one of SEQ ID NOs: 1-57 or 94-148 isapplied, e.g. a polyclonal rabbit antibody. A third antibody thatdetects the second antibody, e.g. an anti-rabbit antibody, conjugated toa suitable label, e.g. an enzyme for chromogenic detection, is thenadded. The plate is then developed with a substrate for the label inorder to detect and quantify the label, which in turn serves as ameasure of any one of SEQ ID NOs: 1-57 or 94-148 in the body fluid. Thisdetermination may then be compared to that of an internal referenceregion measured by similar methodology. If the label is an enzyme forchromogenic detection, the substrate is a color-generating substrate ofthe conjugated enzyme and the color reaction is subsequently detected ina microplate reader and compared to standards.

Suitable pairs of antibodies that may be used as first and secondantibodies are any combination of, e.g., guinea pig, rat, mouse, rabbit,goat, chicken, donkey or horse antibodies. In a particular embodiment,the antibodies are polyclonal antibodies. In another particularembodiment, the antibodies are monoclonal antibodies or antibodyfragments. Suitable labels include: chromogenic labels (enzymes that canbe used to convert a substrate to a detectable colored or fluorescentcompound), spectroscopic labels (e.g., fluorescent labels), and affinitylabels which may be developed by an additional compound specific for thelabel, thereby facilitating detection and quantification, or any otherlabel used in standard ELISA.

Other preferred methods for detection of any one of SEQ ID NOs: 1-57 or94-148 include radioimmunoassay or competitive immunoassay using asingle antibody and chemiluminescence detection on automated commercialanalytical robots. Microparticle enhanced fluorescence, fluorescencepolarized methodologies, or mass spectrometry may also be used.Detection devices, e.g. microarrays, are also useful components asreadout systems for any one of SEQ ID NOs: 1-57 or 94-148.

Also encompassed herein are kits for assessing over-representation orunder-representation of any one of SEQ ID NOs: 1-57 or 94-148 relativeto an internal reference region for determining risk for developingsepsis, which kits may comprise apparatus and reagents for detecting atleast one of SEQ ID NOs: 1-57 or 94-148. Apparatus and reagentsconsidered for PCR amplification include: suitable PCR primer pairsspecific for each of SEQ ID NOs: 1-57 or 94-148, amplification reagents,and thermocycling devices. With respect to ELISA, microtiter plates forELISA, pre-coated ELISA plates, and plate covers are encompassed.Reagents useful for ELISA include those antibodies and solutionsdeveloped and designed for detecting each of SEQ ID NOs: 1-57 or 94-148.Standard solutions comprising each of SEQ ID NOs: 1-57 or 94-148 aspositive controls may be included in such kits. Kits may furthercomprise hardware, such as pipettes, solutions such as buffers, blockingsolutions and the like, filters, and directions for use thereof.

The following definitions are presented as an aid to understand theinvention.

The term “DNA” means a polymer composed of deoxyribonucleotides.

The terms “sample”, “biological sample”, “diagnostic sample”, and thelike refer to a material known or suspected of containing one or morepolynucleotide or polypeptide markers. The diagnostic sample may be anytissue ((e.g., blood, and fractions thereof, including serum, etc.).

The terms “polynucleotide” and “nucleic acid”, used interchangeablyherein, describe a polymer of any length, e.g., greater than about 10bases, greater than about 100 bases, greater than about 500 bases,greater than 1000 bases, usually up to about 10,000 or more basescomposed of nucleotides, such as deoxyribonucleotides orribonucleotides, or compounds produced synthetically which can hybridizewith naturally occurring nucleic acids in a sequence specific manneranalogous to that of two naturally occurring nucleic acids inWatson-Crick base pairing interactions. Polynucleotide and nucleic acidinclude polynucleotides that encode a native-sequence polypeptide, apolypeptide variant, a portion of a polypeptide, a chimeric polypeptide,or an isoform, precursor, complex, modified form, or derivative of anyof the foregoing, and any precursors thereof. Polynucleotides can bedeoxyribonucleotides, ribonucleotides, modified nucleotides or bases,and/or their analogs, or any substrate that can be incorporated into apolymer by DNA or RNA polymerase or by a synthetic reaction. Apolynucleotide may be modified after synthesis (e.g., by conjugationwith a label, such as a radioactive, chemiluminescent, chemiflourescent,or fluorescent label, and the like). Other types of modifications topolynucleotides known to a person skilled in the art includesubstitution of one or more naturally-occurring nucleotides with ananalog, internucleotide modifications (e.g., uncharged linkages, chargedlinkages), and the like.

Polynucleotides can also include circulating nucleic acids (“CNA”). Theterm “circulating nucleic acid” or “CNA” refers to free nucleic acid,including RNA and DNA, circulating in any form in the blood. CNA caninclude gene transcripts, transcription factors or other polynucleotidesequences. CNA can be obtained from any applicable biological sample,including blood, plasma, serum, and the like.

“Variants” of the sequences described herein are sequences wherein atleast one nucleotide differs from that of the native or wild-typesequence (or the complement thereof), by virtue of an insertion,deletion, modification and/or substitution of one or more nucleotideswithin the native sequence. Such variants generally have less than 100%sequence identity relative to a native sequence or its complement.Accordingly, a sequence variant may have a nucleotide sequence with atleast about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequenceidentity relative to the native or wild-type sequence or complementthereof. Variants, furthermore, may include fragments of any length thatretain a biological activity of the corresponding native sequence.Variants also include sequences wherein one or more nucleotides areadded to the 5′ or 3′ end of, or within, a native sequence or itscomplement.

“Percent sequence identity” is defined herein as the percentage ofnucleotides or amino acid residues in the candidate sequence that areidentical to the nucleotides or residues in the sequence of interestafter aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Methods andcomputer programs for the alignment of sequences are well known in theart, including, for example, “BLAST” algorithms.

“Oligonucleotides” include short, single-stranded polynucleotides thatare at least seven nucleotides in length and less than about 250nucleotides in length. The term “polynucleotides” includesoligonucleotides.

“Label” refers to a detectable compound or composition and “labelling”refers to the conjugation, fusion, or attachment of a detectablecompound or composition to another. In some aspects described herein,the label is conjugated or fused directly or indirectly to a reagent,such as a polynucleotide probe or an antibody, and assists with thedetection of the reagent to which it is conjugated or fused. The labelitself can also be detectable (such as radioisotope labels orfluorescent labels and the like). In some aspects described herein, thelabel is an enzymatic label which catalyzes chemical alteration of asubstrate compound or composition and results in a detectable product.

The term “diagnosis”, as used herein, refers to the identification orclassification of a molecular or pathological state, disease, orcondition (e.g., sepsis). In a particular embodiment, sepsis isdiagnosed in a subject (e.g., a human subject) in advance of onset ofsepsis symptoms.

In a particular embodiment, a human subject is characterized as being“at risk for developing sepsis” because they have recently experiencedan accident (e.g., vehicular, bicycle), physical trauma (e.g., due toburns resulting from exposure to, e.g., fire or chemicals; knife orgunshot wounds; blunt force trauma; explosion), surgery, and/orinfection (e.g., a bacterial, fungal, or viral infection). Humansubjects at risk for developing sepsis also include: subjects withweakened immune systems (e.g., immunocompromised subjects); subjectswith pre-existing infections or medical conditions (e.g., type 2diabetes, kidney disease, and asthma), obese subjects, and very youngand very old subjects, and subjects injured in contaminatedenvironments. Assays to evaluate risk for developing sepsis may also beimplemented on all patients admitted to the hospital and/or releasedfrom the hospital as a matter of routine.

In a particular embodiment thereof, each patient released after ahospital stay comprising at least one overnight stay is assayed usingmethods described herein to evaluate whether the patient has early stagesepsis or is at risk for developing sepsis post-release. In that theexpense involved in re-admittance to the hospital is substantial andinsurance coverage is not commensurate with such expenses, screening allpatients released after at least one overnight stay, would be acost-effective way in which to provide better healthcare to the patientand minimize expenditures involved with same.

Further to the above, exemplary indications for use include:

-   -   At hospital admission where the best option today is to observe        symptoms and run a series of blood cultures which normally takes        over 24 hours for results and have high error rates;    -   On high risk patient groups in hospitals where early        identification and treatment of sepsis could have an impact to a        patient's survival;    -   On discharge where ruling out sepsis could lead to early        discharge;    -   In long term care facilities where the frequency of sepsis is        high and early diagnosis and appropriate monitoring and/or        treatment thereafter could potentially reduce costly        re-admissions and/or admissions to hospitals; and    -   Monitoring neonates for neonatal sepsis

Sepsis-3 Definitions

Sepsis is life-threatening organ dysfunction due to a dysregulated hostresponse to infection

Sepsis clinical criteria: organ dysfunction is defined as an increase of2 points or more in the Sequential Organ Failure Assessment (SOFA) score

-   -   for patients with infections, an increase of 2 SOFA points gives        an overall mortality rate of 10%    -   Patients with suspected infection who are likely to have a        prolonged ICU stay or to die in the hospital can be promptly        identified at the bedside with qSOFA (“HAT”); i.e. 2 or more of:        -   Hypotension: SBP less than or equal to 100 mmHg        -   Altered mental status (any GCS less than 15)        -   Tachypnoea: RR greater than or equal to 22.

Septic shock is a subset of sepsis in which underlying circulatory andcellular/metabolic abnormalities are profound enough to substantiallyincrease mortality

-   -   Septic shock clinical criteria: Sepsis and (despite adequate        volume resuscitation) both of:        -   Persistent hypotension requiring vasopressors to maintain            MAP greater than or equal to 65 mm Hg, and        -   Lactate greater than or equal to 2 mmol/l,    -   With these criteria, hospital mortality is in excess of 40%.

“Primer” refers to a polynucleotide capable of acting as a point ofinitiation of synthesis along a complementary strand when conditions aresuitable for synthesis of a primer extension product. The synthesizingconditions include the presence of four different nucleotide bases(adenosine, cytidine, guanosine, thymidine/uridine) and at least onepolymerization-inducing agent such as a reverse transcriptase or a DNApolymerase. The primers are present in a suitable buffer, which mayinclude constituents which are co-factors or affect conditions such aspH and the like at various suitable temperatures. Primer includessingle-stranded polynucleotide that is capable of hybridizing to nucleicacid and allowing the polymerization of a complementary nucleic acid,generally by providing a free 3′-OH group. Double stranded sequences canalso be utilized. Primers are typically at least about 15 nucleotides.In some embodiments, primers can have a length of from about 15 to about30, about 15 to about 50, about 15 to about 75, about 15 to about 100,or about 15 to about 500 nucleotides.

Exemplary primer pairs specific for each of SEQ ID NOs: 1-57 or 94-148are presented herein in Table 1 or Table 3. Exemplary primer pairsspecific for each of SEQ ID NOs: 58-93 are presented herein in Table 2.Such primer pairs are selected based on their specificity for aparticular polynucleotide and may be optimized for use in connectionwith, e.g., PCR amplification. Polynucleotide-specific primer pairs maycomprise primers that include variations within their sequence such thatthe primer is no longer 100% complementary to the polynucleotide forwhich it is specific. Primers comprising such variations are encompassedherein as long as the variations do not alter the ability of the primerto amplify the polynucleotide with specificity. Such variations may alsoinclude nucleotides and/or tags at the 5′ and 3′ ends of the primer thatare not complementary the polynucleotide for which the primer isspecific. It is also understood that a primer or primer pair may becomplementary to sequences that flank any one of SEQ ID NOs: 1-148 inthe human genome and thus, may be used to amplify one of SEQ ID NOs:1-148 in keeping with methods described herein. Design of such primersand primer pairs is well within the capabilities of one of ordinaryskill in the art having read the present specification.

In a particular embodiment, multiplex PCR based on the TaqMan approachis utilized to detect CNAs of interest. Such embodiments call for aprimer pair, wherein each of the primers is specific for the particularCNA (polynucleotide) of interest and one labeled probe which is specificfor the particular CNA (polynucleotide) of interest and labeled at eachterminus with a different moiety. In a particular embodiment, thedifferent moiety can be a fluorophore at the first terminus and aquencher at the second terminus. Such fluorophore-quencher pairscomprise, for example, FAM-BHQ1, HEX-BHQ1, LC610-BHQ2, CY5-BHQ-2, andCY5.5-BBQ650. Other pairs useful for this embodiment are known in theart.

A “motif” or “sequence motif” refers to a nucleotide sequence patternthat is generally conserved across multiple species. Polynucleotides canbe derived from the motif. The polynucleotides can correspond to theentire sequence of the motif or a portion or portions of the motif.

“Marker” or “biomarker” refers to an indicator which can be detected ina sample, and includes predictive, diagnostic, and prognostic indicatorsand the like. The marker can be an indicator of a particular disease ordisorder (e.g., sepsis) having certain molecular, pathological,histological, and/or clinical features. Exemplary biomarkers include,without limitation, polynucleotides, polypeptides, polypeptide andpolynucleotide modifications (such as post-translational modificationsand the like), carbohydrates, and/or glycolipid-based molecular markers.The “presence”, “amount”, or “level” of a marker associated with anincreased clinical benefit to an individual is a detectable level of themarker in a sample. The presence, amount, or level of a marker can bemeasured by methods known to a person skilled in the art. The presence,amount, or level of a marker may be measured prior to treatment, duringtreatment, after treatment, or a combination of any of the foregoing.

“Internal reference region” refers to a nucleic acid fragmentcirculating in a bodily fluid (e.g., blood or a fraction thereof such asserum) that is present in the same amount in both control subjects(those subjects who do not have sepsis, those subjects who are not atrisk for developing sepsis, and/or those subjects who do not developsepsis in three days) and subjects who have sepsis, as determined, forexample, by RT-PCR experiments. Internal reference regions provide anucleic acid fragment which is represented in the bodily fluid at aparticular level, against which representation of other nucleic acidsequences (e.g., any one of SEQ ID NOs: 1-57 or 94-148), which differ incontrol subjects and sepsis patients and are therefore discriminatory,can be evaluated in a relative manner. An internal reference system canbe at least one region but can also be a composition of a few regions.

“Over-representation” refers to a fold increase (relative quantity RQ)relative to the internal reference region calculated as at least2^(−delta-delta Cq) of 2.

“Under-representation” refers to a fold decrease (relative quantity RQ)relative to the internal reference region calculated as at most2^(−delta-delta Cq) of 0.5.

The ΔCq method normalizes disease-specific motifs within a sample bysubtracting the Cq value of the internal reference region (referencemotif) from the Cq value of the disease specific motifs.

The 2−ΔΔCq method calculates relative quantity (RQ) of normalizeddisease specific motifs between two sample types (i.e., healthy anddiseased). Information from multiple reference motifs can be combined toimprove accuracy.

In a PCR reaction, the Cq (Cycle Quantification) value is the PCR cyclenumber at which the sample's reaction curve intersects the thresholdline. This value tells how many cycles it took for the PCR machine todetect a real signal from the sample. Therefore, lower Cq valuesindicate higher amounts of target nucleic acid fragments, whereas higherCq values indicate lower amounts of target nucleic acid fragments.

Exemplary CNAs identified herein, which are markers of sepsis, includeSEQ ID NOs: 1-57 and 94-148. See Tables 1 and 3 (FIGS. 1 and 3).Exemplary primer pairs for amplifying each of SEQ ID NOs: 1-57 and94-148 are also presented in Tables 1 and 3 (FIGS. 1 and 3). Referenceis made to the chromosomal locations to which each of these CNAs can befound in the human genome. Accordingly, longer polynucleotidescomprising each of SEQ ID NOs: 1-57 and 94-148, respectively, canreadily be determined via access to publicly available databases.Additional primers for amplifying each of SEQ ID NOs: 1-57 and 94-148,respectively, can be designed based on the availability of suchadditional sequence information.

Exemplary subsets of SEQ ID NOs: 1-57 and 94-148 comprise at least two,at least three, at least four, at least five, at least six, at leastseven, at least eight, at least nine, at least ten, at least eleven, atleast twelve, at least thirteen, at least fourteen, at least fifteen, atleast sixteen, at least seventeen, at least eighteen, at least nineteen,at least twenty, at least twenty-one, at least twenty-two, at leasttwenty-three, or at least twenty-four of the polynucleotides. Exemplarysubsets and primers for amplification of each of the markers offerreagents for detecting CNAs comprising the indicated SEQ ID NOs:. SeeTables 1-3. In a particular embodiment, detecting CNAs comprising theindicated SEQ ID NOs: is indicative of sepsis.

An exemplary subset may be provided in a kit for detecting at least oneof SEQ ID NOs: 1-148 and subsets thereof (see, e.g., Table 4).

Table 4 presents an exemplary subset comprising, consisting essentiallyof, and/or consisting of the indicated SEQ ID NOs: 1-2, 4-6, 30-31, 38,44, 46, 50, 53, 55, 59, 61, 68, 100, 108-110, 112, 114, 116, and 129.Primers for amplification of each of the markers listed in Table 4 canbe found in the corresponding Tables 1, 2, or 3. Also encompassed hereinare kits comprising the 24 exemplary markers presented in Table 4 andsubsets thereof.

TABLE 4 List of 24 Exemplary Markers Listed in Final motif ID SEQ ID NO:Patent ID Table 1 Sepsis JC1 1 hu-sep-CNAD-0001 Table 1 Sepsis JC2 2hu-sep-CNAD-0002 Table 1 Sepsis JC4 4 hu-sep-CNAD-0004 Table 1 SepsisJC5 5 hu-sep-CNAD-0005 Table 1 Sepsis JC6 6 hu-sep-CNAD-0006 Table 1Sepsis JC34 30 hu-sep-CNAD-0030 Table 1 Sepsis JC35 31 hu-sep-CNAD-0031Table 1 Sepsis JC42 38 hu-sep-CNAD-0038 Table 1 Sepsis JC48 44hu-sep-CNAD-0044 Table 1 Sepsis JC50 46 hu-sep-CNAD-0046 Table 1 SepsisSC2 50 hu-sep-CNAD-0050 Table 1 Sepsis SC5 53 hu-sep-CNAD-0053 Table 1Sepsis SC7 55 hu-sep-CNAD-0055 Table 2 Sepsis JU2 59 hu-sep-CNAD-0059Table 2 Sepsis JU4 61 hu-sep-CNAD-0061 Table 2 Sepsis JU11 68hu-sep-CNAD-0068 Table 3 Sepsis J7 100 hu-sep-CNAD-0100 Table 3 SepsisJ15 108 hu-sep-CNAD-0108 Table 3 Sepsis J16 109 hu-sep-CNAD-0109 Table 3Sepsis J17 110 hu-sep-CNAD-0110 Table 3 Sepsis J19 112 hu-sep-CNAD-0112Table 3 Sepsis J21 114 hu-sep-CNAD-0114 Table 3 Sepsis J23 116hu-sep-CNAD-0116 Table 3 Sepsis J36 129 hu-sep-CNAD-0129

Exemplary CNAs identified herein, which are internal reference regions,include SEQ ID NOs: 58-93. See Table 2 (FIG. 2). Exemplary primer pairsfor amplifying each of SEQ ID NOs: 58-93 are also presented in Tables1-3 (FIG. 1-3). Reference is made to the chromosomal locations to whicheach of these CNAs can be found in the human genome. Accordingly, largerpolynucleotides comprising each of SEQ ID NOs: 58-93, respectively, canreadily be determined via access to publicly available databases.Additional primers for amplifying each of SEQ ID NOs: 58-93,respectively, can be designed based on the availability of suchadditional sequence information.

Candidate internal reference regions (Table 2: SEQ ID NOs: 58-93) wereevaluated to identify the ones that were most consistently representedacross a set of sepsis and control samples. Given a candidate referenceregion, the differences in median Cq values of the regions betweensepsis samples and control samples were calculated. The lesser thedifference in the median Cq values, the higher the consistency of theregion across sepsis and non-sepsis conditions, and therefore the moresuitable the region as an internal reference region.

The three most consistent regions were selected as exemplary internalreference regions (SEQ ID NOs: 59, 61 and 68). Their sequences andprimers used for amplification are listed in Table 2 in thecorresponding rows of the regions.

An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ IDNOs: 1, 6, 46, 50, 53, 55, 100, 109, 110, 114, and at least one of SEQID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 1, 6, 46, 50,53, 55, 59, 61, 68, 100, 109, 110, and 114 are presented in Tables 1-3.

An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ IDNOs: 1, 2, 6, 30, 53, 55, 110, 112, 114, 116, and at least one of SEQ IDNOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 1, 2, 6, 30, 53,55, 59, 61, 68, 110, 112, 114, and 116 are presented in Tables 1-3.

An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ IDNOs: 30, 31, 38, 44, 46, 50, 53, 55, 100, 129, and at least one of SEQID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 30, 31, 38,44, 46, 50, 53, 55, 59, 61, 68, 100, and 129 are presented in Tables1-3.

An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ IDNOs: 4, 5, 30, 31, 100, 108, 109, 110, 116, 129, and at least one of SEQID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 4, 5, 30, 31,59, 61, 68, 100, 108, 109, 110, 116, and 129 are presented in Tables1-3.

An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ IDNOs: 53, 55, 100, 108, 109, 110, 112, 114, 116, 129, and at least one ofSEQ ID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 53, 55,59, 61, 68, 100, 108, 109, 110, 112, 114, 116, and 129 are presented inTables 1-3.

An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ IDNOs: 30, 31, 46, 55, 108, 110, 112, 114, 116, 129, and at least one ofSEQ ID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 30, 31,46, 55, 59, 61, 68, 108, 110, 112, 114, 116, and 129 are presented inTables 1-3.

An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ IDNOs: 1, 2, 5, 46, 53, 100, 112, 114, 116, 129, and at least one of SEQID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 1, 2, 5, 46,53, 59, 61, 68, 100, 112, 114, 116, and 129 are presented in Tables 1-3.

An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ IDNOs: 2, 4, 6, 31, 44, 100, 109, 110, 116, 129 and at least one of SEQ IDNOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 2, 4, 6, 31, 44,59, 61, 68, 100, 109, 110, 116, and 129 are presented in Tables 1-3.

An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ IDNOs: 5, 6, 30, 31, 44, 50, 55, 100, 110, 112, and at least one of SEQ IDNOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 5, 6, 30, 31, 44,50, 55, 59, 61, 68, 100, 110, and 112 are presented in Tables 1-3.

An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ IDNOs: 2, 31, 38, 44, 46, 55, 109, 110, 112, 129, and at least one of SEQID NOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 2, 31, 38, 44,46, 55, 59, 61, 68, 109, 110, 112, and 129 are presented in Tables 1-3.

An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ IDNOs: 4, 5, 6, 30, 38, 44, 53, 55, 108, 109, and at least one of SEQ IDNOs: 59, 61, or 68. Primers for amplifying SEQ ID NOs: 4, 5, 6, 30, 38,44, 53, 55, 59, 61, 68, 108, and 109 are presented in Tables 1-3.

An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ IDNOs: 31, 46, 55, 108, 114, and at least one of SEQ ID NOs: 59, 61, or68. Primers for amplifying SEQ ID NOs: 31, 46, 55, 59, 61, 68, 108, and114 are presented in Tables 1-3.

An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ IDNOs: 100, 109, 112, 114, 116, and at least one of SEQ ID NOs: 59, 61, or68. Primers for amplifying SEQ ID NOs: 59, 61, 68, 100, 109, 112, 114,and 116 are presented in Tables 1-3.

An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ IDNOs: 31, 38, 44, 100, 108, and at least one of SEQ ID NOs: 59, 61, or68. Primers for amplifying SEQ ID NOs: 31, 38, 44, 59, 61, 68, 100, and108 are presented in Tables 1-3.

An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ IDNOs: 1, 4, 5, 110, 112, and at least one of SEQ ID NOs: 59, 61, or 68.Primers for amplifying SEQ ID NOs: 1, 4, 5, 59, 61, 68, 110, and 112 arepresented in Tables 1-3.

An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ IDNOs: 5, 6, 44, 55, 109, and at least one of SEQ ID NOs: 59, 61, or 68.Primers for amplifying SEQ ID NOs: 5, 6, 44, 55, 59, 61, 68, and 109 arepresented in Tables 1-3.

An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ IDNOs: 1, 2, 5, 50, 53, and at least one of SEQ ID NOs: 59, 61, or 68.Primers for amplifying SEQ ID NOs: 1, 2, 5, 50, 53, 59, 61, and 68 arepresented in Tables 1-3.

An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ IDNOs: 4, 5, 6, 31, 38, and at least one of SEQ ID NOs: 59, 61, or 68.Primers for amplifying SEQ ID NOs: 4, 5, 6, 31, 38, 59, 61, and 68 arepresented in Tables 1-3.

An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ IDNOs: 1, 53, 100, 114, 116, and at least one of SEQ ID NOs: 59, 61, or68. Primers for amplifying SEQ ID NOs: 1, 53, 59, 61, 68, 100, 114 and116 are presented in Tables 1-3.

An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ IDNOs: 5, 6, 30, 31, 38, and at least one of SEQ ID NOs: 59, 61, or 68.Primers for amplifying SEQ ID NOs: 5, 6, 30, 31, 38, 59, 61 and 68 arepresented in Tables 1-3.

An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ IDNOs: 44, 46, 50, 53, 55, and at least one of SEQ ID NOs: 59, 61, or 68.Primers for amplifying SEQ ID NOs: 44, 46, 50, 53, 55, 59, 61, and 68are presented in Tables 1-3.

An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ IDNOs: 109, 110, 112, 114, 116, and at least one of SEQ ID NOs: 59, 61, or68. Primers for amplifying SEQ ID NOs: 59, 61, 68, 109, 110, 112, 114,and 116 are presented in Tables 1-3.

An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ IDNOs: 4, 5, 114, 116, 119 and at least one of SEQ ID NOs: 59, 61, or 68.Primers for amplifying SEQ ID NOs: 4, 5, 59, 61, 68, 114, 116, and 119are presented in Tables 1-3.

An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ IDNOs: 4, 6, 30, 100, 108 and at least one of SEQ ID NOs: 59, 61, or 68.Primers for amplifying SEQ ID NOs: 4, 6, 30, 59, 61, 68, 100, and 108are presented in Tables 1-3.

An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ IDNOs: 38, 44, 109, 110, 112 and at least one of SEQ ID NOs: 59, 61, or68. Primers for amplifying SEQ ID NOs: 38, 44, 59, 61, 68, 109, 110, and112 are presented in Tables 1-3.

An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ IDNOs: 1, 2, 4, 5, and at least one of SEQ ID NOs: 59, 61, or 68. Primersfor amplifying SEQ ID NOs: 1, 2, 4, 5, 59, 61, and 68 are presented inTables 1-3.

An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ IDNOs: 30, 53, 108, 109, and at least one of SEQ ID NOs: 59, 61, or 68.Primers for amplifying SEQ ID NOs: 30, 53, 59, 61, 68, 108, and 109 arepresented in Tables 1-3.

An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ IDNOs: 2, 109, 110, 116, and at least one of SEQ ID NOs: 59, 61, or 68.Primers for amplifying SEQ ID NOs: 2, 59, 61, 68, 109, 110, and 116 arepresented in Tables 1-3.

An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ IDNOs: 5, 6, 50, 53, and at least one of SEQ ID NOs: 59, 61, or 68.Primers for amplifying SEQ ID NOs: 5, 6, 50, 53, 59, 61, and 68 arepresented in Tables 1-3.

An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ IDNOs: 1, 31, 55, 108, and at least one of SEQ ID NOs: 59, 61, or 68.Primers for amplifying SEQ ID NOs: 1, 31, 55, 59, 61, 68, and 108 arepresented in Tables 1-3.

An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ IDNOs: 30, 38, 55, 100, and at least one of SEQ ID NOs: 59, 61, or 68.Primers for amplifying SEQ ID NOs: 30, 38, 55, 59, 61, 68, and 100 arepresented in Tables 1-3.

An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ IDNOs: 2, 4, 112, 114, and at least one of SEQ ID NOs: 59, 61, or 68.Primers for amplifying SEQ ID NOs: 2, 4, 59, 61, 68, 112, and 114 arepresented in Tables 1-3.

An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ IDNOs: 31, 110, 112, 114, and at least one of SEQ ID NOs: 59, 61, or 68.Primers for amplifying SEQ ID NOs: 31, 59, 61, 68, 110, 112, and 114 arepresented in Tables 1-3.

An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ IDNOs: 5, 31, 116, 129, and at least one of SEQ ID NOs: 59, 61, or 68.Primers for amplifying SEQ ID NOs: 5, 31, 59, 61, 68, 116, and 129 arepresented in Tables 1-3.

An exemplary subset of SEQ ID NOs: set forth in Table 4 comprises SEQ IDNOs: 2, 4, 5, 6, and at least one of SEQ ID NOs: 59, 61, or 68. Primersfor amplifying SEQ ID NOs: 2, 4, 5, 6, 59, 61, and 68 are presented inTables 1-3.

Machine learning classification systems, such as, e.g., a support vectoror neural network are utilized herein. The system utilized hereincreates a multidimensional ‘map’ comprising basically all the markers(both sepsis and non-sepsis) and then when a sample is tested, it'sposition on the map it marked. After this, the system looks at theclosest points and if they are sepsis, it will call the sample ‘sepsis’.If the closest points are not sepsis, it will call the sample ‘notsepsis’. In a particular embodiment, the system looks for a certainsmall number of neighboring samples (not the single closest one) in thisvirtual multidimensional space, where each sample is represented by agroup of marker values on the sample. The assignment of the sample tosepsis/non-sepsis is accomplished by a majority vote of the known labelsof the closest neighbors (e.g., 5 of them). If there are more sepsissamples in the neighborhood, the incoming sample is assigned to“sepsis”. If there are more non-sepsis samples in the neighborhood, thenew sample is assigned to “non-sepsis”.

“Encode” refers to a polynucleotide “encoding” a polypeptide if, in itsnative state or when manipulated by methods well known to those skilledin the art, it can be transcribed and/or translated to produce the mRNAfor the polypeptide and/or the polypeptide (or a fragment thereof). Theanti-sense strand is the complement of such a nucleic acid, and theencoding sequence can be deduced therefrom.

“Array” or “microarray” refers to an ordered arrangement of hybridizablearray elements on a substrate, such as a solid substrate (e.g., glassslide and the like) or a semi-solid substrate (e.g., nitrocellulosemembrane and the like). In some embodiments, the array elements may bepolynucleotide probes (e.g. oligonucleotide). Arrays may include DNAmicroarrays (including cDNA microarrays, oligonucleotide microarrays,SNP microarrays, etc.), protein microarrays, peptide microarrays,antibody microarrays, and the like.

“Amplification” or “amplifying” refers to the production of one or morecopies of a reference nucleic acid sequence or its complement.Amplification may be linear or exponential (e.g., in a polymerase chainreaction (PCR)). A nucleic acid copy produced from amplification may nothave perfect sequence complementarity or identity relative to thereference sequence. In some embodiments, the copies can includenucleotide analogs, including deoxyinosine, intentional sequencealterations (such as alterations introduced through a primer that ishybridizable, but not fully complementary, to the template), and/orsequence errors that occur during the amplification process.

The terms “expression” and “expression level”, in general, are usedinterchangeably and generally refer to the amount of a marker in asample. “Expression” generally refers to the process by whichinformation (e.g., gene-encoded and/or epigenetic) is converted into thestructures present and operating in the cell. Therefore, as used herein,“expression” can refer to transcription into a polynucleotide (such asmRNA and the like), translation into a polypeptide, or evenpolynucleotide and/or polypeptide modifications (e.g.,post-translational modification of a polypeptide and the like).Fragments of the transcribed polynucleotide, the translated polypeptide,or polynucleotide and/or polypeptide modifications (such aspost-translational modification of a polypeptide and the like) will alsobe regarded as expressed whether they originate from a transcriptgenerated by alternative splicing or a degraded transcript, or from apost-translational processing of the polypeptide (e.g., by proteolysis).“Expressed genes” include those that are transcribed into apolynucleotide as mRNA and then translated into a polypeptide, and alsothose that are transcribed into RNA but not translated into apolypeptide (such as transfer and ribosomal RNAs and the like).

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule. The nucleic acid moleculemay be present extrachromosomally or at a chromosomal location that isdifferent from its natural location.

The term “sequencing” and its variants include obtaining sequenceinformation from a strand of a nucleic acid molecule, typically bydetermining the identity of at least some nucleotides (including theirnucleic acid components) within the nucleic acid molecule. The termsequencing may also refer to determining the order of nucleotides (basesequences) in a nucleic acid sample (e.g. DNA or RNA). Many techniquesare available and known to a person skilled in the art, such as Sangersequencing, high-throughput sequencing technologies (such as the GS FLXplatform formerly offered by Roche Applied Science, Penzberg, Germany,based on pyro sequencing, or Illumina sequencing platforms, as offeredby Illumina Inc., 5200 Illumina Way, San Diego, Calif. 92122, USA), andthe like. High-throughput sequencing technologies refer to sequencingtechnologies having increased throughput as compared to traditionalSanger- and capillary-electrophoresis-based approaches (e.g., with theability to generate hundreds of thousands or millions of relativelysmall sequence reads at a time). High throughput sequencing technologiesinclude, but are not limited to, sequencing by synthesis, sequencing byligation, pyrosequencing, sequencing by hybridization, and/or the like.

As used herein, “reactive” means the agent has affinity for, binds to,or is directed against a specific CNA. As further used herein, an“agent” includes a protein, polypeptide, peptide, nucleic acid(including DNA or RNA), antibody, Fab fragment, F(ab′)2 fragment,molecule, compound, antibiotic, drug, and any combinations thereof. AFab fragment is a univalent antigen-binding fragment of an antibody,which is produced by papain digestion. A F(ab′)2 fragment is a divalentantigen-binding fragment of an antibody, which is produced by pepsindigestion. By way of example, the agent of the present invention can belabeled with a detectable marker. Agents that are reactive with CNAs canbe identified by contacting the CNA with an agent of interest andassessing the ability of the agent to bind to the CNA.

In one embodiment of the present invention, the agent reactive with asepsis biomarker is an antibody. Antibodies for use herein can belabeled with a detectable marker. Labeling of an antibody can beaccomplished using one of a variety of labeling techniques, includingperoxidase, chemiluminescent labels known in the art, and radioactivelabels known in the art. The detectable marker of the present inventioncan be, for example, a nonradioactive or fluorescent marker, such asbiotin, fluorescein (FITC), acridine, cholesterol, orcarboxy-X-rhodamine (ROX), which can be detected using fluorescence andother imaging techniques readily known in the art. Alternatively, thedetectable marker can be a radioactive marker, including, for example, aradioisotope. The radioisotope can be any isotope that emits detectableradiation, such as ³⁵S, ³²P, or ³H. Radioactivity emitted by theradioisotope can be detected by techniques well known in the art. Forexample, gamma emission from the radioisotope can be detected usinggamma imaging techniques, particularly scintigraphic imaging. By way ofexample, the agent of the present invention is a high-affinity antibodylabeled with a detectable marker.

Where the agent of the present invention is an antibody reactive with asepsis biomarker, a biological sample taken from a mammal (e.g., ahuman) can be purified by passage through an affinity column whichcontains the antibody having affinity to the sepsis biomarker as aligand attached to a solid support, such as an insoluble organic polymerin the form of a bead, gel, or plate. The antibody attached to the solidsupport can be used in the form of a column. Examples of suitable solidsupports include, without limitation, agarose, cellulose, dextran,polyacrylamide, polystyrene, sepharose, and other insoluble organicpolymers. The antibody can be further attached to the solid supportthrough a spacer molecule, if desired. Appropriate binding conditions(e.g., temperature, pH, and salt concentration) can be readilydetermined by the skilled artisan. By way of example, the antibody canbe attached to a sepharose column, such as Sepharose 4B.

Alternatively, a biological sample from a mammal (e.g., a human) can beassayed using hybridization analysis of nucleic acid extracted from thebiological sample taken from the mammal (e.g., a human) to determine thepresence of a sepsis biomarker, such as a CNA. This method also can beconducted by performing a Southern blot analysis of DNA using at leastone nucleic acid probe which hybridizes to CNAs (including amplifiedCNAs). The nucleic acid probes described herein can be prepared by avariety of techniques known to those skilled in the art, including,without limitation, the following: restriction enzyme digestion ofnucleic acid; and automated synthesis of oligonucleotides havingsequences which correspond to selected portions of the nucleotidesequence of the sepsis biomarker, using commercially-availableoligonucleotide synthesizers.

The nucleic acid probes used herein can be DNA or RNA, and can vary inlength from about 5-20 nucleotides or 10-20 nucleotides to the entirelength of the nucleic acid encoding for a sepsis biomarker. In someembodiments, the nucleic acid probes are oligonucleotides. The nucleicacid used in the probes can be derived from mammalian polynucleotidesequence complementary to the sepsis biomarker. In addition, the nucleicacid probes of the present invention can be labeled with one or moredetectable markers. Labeling of the nucleic acid probes can beaccomplished using one of a number of methods known in the art (e.g.,nick translation, end labeling, fill-in end labeling, polynucleotidekinase exchange reaction, random priming, SP6 polymerase (for riboprobepreparation)) along with one of a variety of labels (e.g., radioactivelabels, such as ³⁵S, ³²P, or ³H, or nonradioactive labels, such asbiotin, fluorescein (FITC), acridine, cholesterol, orcarboxy-X-rhodamine (ROX)). In some embodiments, these nucleic acidprobes are used in an array or microarray.

In addition, the present invention provides a method of determiningwhether a human has sepsis, but is asymptomatic, or has alreadydeveloped sepsis and has symptoms thereof. The method includes analyzinga biological sample of the human for the presence of at least one sepsisbiomarker, and optionally, further recommending a corroborative test forsepsis if the at least one sepsis biomarker is present in the biologicalsample. In some embodiments, the corroborative test includes ELISA,immunohistochemistry, and Western Blot/immunoblot or a combination ofmore than one of any of the foregoing.

In the methods described herein, the step of analyzing a biologicalsample may optionally include obtaining the sample from the human;isolating nucleic acid from the sample; amplifying the isolated nucleicacid using primers that are specific for or capable of amplifying asequence corresponding to a sepsis CNA biomarker; and sequencing theamplified nucleic acid. In some embodiments, the isolated nucleic acidincludes genomic DNA, mRNA, and/or cDNA obtained from mRNA. In someembodiments, the step of determining representation of the at least onesepsis CNA marker includes use of at least one of a PCR-based detectionmethod and a hybridization-based method. In some embodiments, the stepof determining representation of the at least sepsis CNA marker includesan immunohistochemical analysis. In some embodiments, an array or amicroarray is used for identifying the sepsis biomarker.

The biological sample can be assayed for expression of sepsis biomarkersin vitro or in vivo. In addition, the biological sample can be assayedfor expression of sepsis biomarkers using all of the various assays andmethods of detection and quantification described above.

The discovery that certain CNAs constitute sepsis biomarkers providescompositions and methods for identifying a human having early stagesepsis (e.g., pre-symptomatic sepsis), and presents the potential forcommercial application in the form of a test for the diagnosis of sepsisand kits including same. The development of such a test or kit wouldprovide general screening procedures; these procedures could assist inthe early detection and diagnosis of sepsis in human subjects.Accordingly, the present invention further provides a kit for use as anassay of sepsis, comprising at least one agent reactive with a sepsisbiomarker. The agent can be any of those described above, and can beused in any of the above-described assays or methods for detectingsepsis biomarkers.

Oligonucleotides complementary to a sepsis CNA biomarker can be designedbased on the nucleotide sequence of the particular CNA sepsis biomarker.A nucleotide sequence complementary to the selected partial sequence ofthe sepsis CNA biomarker can, e.g., be chemically synthesized using oneof a variety of techniques known to those skilled in the art, including,without limitation, automated synthesis of oligonucleotides havingsequences which correspond to a partial sequence of the sepsis CNAbiomarker nucleotide sequence, or a variation sequence thereof, usingcommercially-available oligonucleotide synthesizers.

The present invention also provides the use of an oligonucleotidecapable of identifying at least one sepsis CNA biomarker to determinethe representation of same in a human. The oligonucleotide can belabelled with a detectable marker, such as a radioactive marker,fluorescent marker, the like, or a combination of any of the foregoing.

Serum from healthy individuals (controls) and diseased patients (i.e.sepsis patients) was harvested and initially stored at −80 degreesCelsius. Total DNA was extracted from the serum samples using the HighPure Viral Nucleic Acid Kit (Roche Applied Science; Cat. No.11858874001). The DNA was amplified using the GenomePlex Single CellWhole Genome Amplification Kit (Sigma; Cat. No. WGA4-500RXN) andpurified using the GenElute™ PCR Clean-Up Kit (Sigma, Cat. No.NA1020-1KT). High-throughput paired-end DNA sequencing was performed bySEQ-IT Kaiserslautern Sequencing facility on NextSeq 500 sequencer andSEQ-IT machines. The resulting sequence reads were mapped to the humangenome. Using an in-house Bioinformatics pipeline, which was establishedon the high-performance Bioinformatics infrastructure at the Instituteof Computational Biotechnology at the Technical University of Graz,Austria, the present inventors have identified DNA motifs, which werepresent at different read count numbers in diseased humans, whencompared to controls. The motifs identified are the result of the hostresponse (i.e., the response of the human body to sepsis). The DNAmotifs identified were used as targets for the development of areal-time Polymerase-Chain Reaction (RT-PCR) assay. In addition to themotifs, which are present at different levels in healthy controls andsepsis patients (markers; SEQ ID NOs: 1-57 or 94-148), motifs which werepresent at the same level in healthy and diseased humans were alsoidentified to allow the normalization of the results (SEQ ID NOs:58-93). The RT-PCR-evaluated motifs, which could be used to discriminatein a statistically significant manner between healthy controls andsepsis patients, stand-alone, or in combination with each other, are thesubject basis for assays described herein.

In a particular embodiment, the Illumina sequences were analyzed using agenome assembly/mapping bioinformatics processing method. The cleanedsequence reads were mapped to the human genome using a fast mapper,genomic hotspots for sepsis were identified, the reads werecomprehensively mapped to the identified hotspots, and a gene search wasconducted in the hotspot region to correlate the reads with a gene.Reads that did not map to the genome were assembled to generate clustersand then analyzed using the same procedures used for the hotspots.

In an embodiment of an Illumina sequence analysis method, CNA librariesare obtained from a blood sample collection and these libraries aresequenced in an Illumina sequencing operation. After the sequencingoperation, the method proceeds to an operation step, where theprocessing unit stores the output of the Illumina sequencing in atext-based file format, for example, a FASTQ format. It is understoodthat the output of the Illumina sequencing can also be stored in otherfile formats, such as, for example, SAM or BAM formats. The sequencingfiles are assembled with a reference genome to determine genomelocations with significant counts of exclusively at risk or normal (notat risk) reads.

The results of the alignment are then processed. In some embodiments,the results of the alignment process are outputted in a text-based fileformat, such as a SAM format. A SAM formatted file is a tab-delimitedtext file that contains sequence alignment data. In such embodiments,the processing unit converts the SAM format to a BAM format, which is abinary version of the SAM formatted file. In other examples, thealignment process outputs a BAM formatted file, and thus, the conversionstep is skipped. Preparation for and creation of an index fileassociated with the BAM formatted file is then created. The index filecreation process may include categorizing by adding sample names as readgroups, sorting, and/or merging.

The created index file and the BAM formatted file is further processedby a processing unit. In particular, processing includes reviewing thealignments and extracting the alignments, which have sufficient coveragefor each contiguous sequence (=contig) of the reference genome. In anembodiment, the alignment parameters comprise a bucket size of 25 and aminimum coverage of 5, although it is understood that the bucket sizeand minimum coverage value may differ in alternate embodiments. Controlregions and at risk regions having 50% or more proportion are extractedand compared to determine overlaps. In some embodiments, these regionsare stored in a database associated with the processing unit for laterretrieval and/or review. Next, the extracted control regions aresubtracted from the at risk regions so that only the at risk regionsremain, thereby providing sequence reads that are present in only atrisk regions. In an embodiment, the control filtering parameterscomprise a minimum subjects value of 3 and a minimum proportion value of0.5, although it is understood that these values may differ in alternateembodiments.

In some embodiments, not all of the sequences are aligned. In suchexamples, the unaligned sequences are collected and may be stored in thedatabase for later retrieval and/or review. In an embodiment, theunaligned sequences are aligned against other references, such as viralreferences, to determine if any alignments exist. If alignments exist,the sequences may be stored in the database for later retrieval andalignment with new viruses, as desired.

In an embodiment, analysis of Illumina sequencing reads can be achievedusing a TimeLogic® Decypher® biocomputing platform (Active Motif,Carlsbad, Calif.) and multiple CPU servers at the sequencing center.

Sequence variability of the identified CNA motifs specific to sepsisrisk was then analyzed in a sampling of different humans through PCR andSanger sequencing of the PCR products to determine if the identified CNAmotifs are capable of detecting sepsis risk generally. In healthyhumans, it has been shown that approximately 97% of CNA sequences are ofgenomic origin [Beck et al., 2009, Clin Chem., 55(4):730-8].

Arrays comprising one of more polynucleotides of the disclosure, PCRprimers and/or probes for amplifying and/or detecting polynucleotides(CNAs) described herein, and methods for detecting risk for developingsepsis comprising an array or PCR primers and/or probes are encompassedherein.

One of more polynucleotide sequences of the disclosure can beincorporated onto a sequence array, such as a biochip, DNA chip,BiofireDX filmarray and other filmarrays, microarray, macroarray, andthe like, for screening, e.g., serum separated from whole blood fromhumans for sepsis risk. Alternatively, CNAs can be extracted from thesample for screening on the array. Arrays are generally solid supportsupon which a collection of polynucleotides and/or primers and/or probesare placed at defined locations on the array, either by spotting,printing, or direct synthesis. The array can include probescorresponding to one or more of the polynucleotides described herein(e.g., at least one of SEQ ID NOs: 1-57 or 94-148 and/or primers and/orprobes for amplifying and/or detecting one or more polynucleotides ofSEQ ID NOs: 1-57 or 94-148 and at least one of SEQ ID NOs: 59, 61, or 68and/or primers and/or probes for amplifying and/or detecting one or morepolynucleotides of SEQ ID NOs: 59, 61, or 68).

The underlying principle of arrays is base pairing or hybridizationi.e., A-T and G-C for DNA, and A-U and G-C for RNA. A sample from amammal (e.g., a human) is allowed to hybridize with the polynucleotidesand/or primers and/or probes on the array providing an expressionprofile/pattern of CNA. The CNA expression pattern of sepsis-specificsequences can be used to determine if a human has early stage sepsis.The array can be prepared by any method known in the art. In someembodiments, a microarray is prepared generally as disclosed in U.S.Pat. No. 7,655,397, the entirety of which is hereby incorporated byreference.

In some embodiments, the array comprises at least 2 polynucleotidesselected from polynucleotides comprising or consisting of SEQ ID NOs:1-57 or 94-148 or primers or probes specific for at least 2 of SEQ IDNOs: 1-57 or 94-148. In some embodiments, the array comprises at least 4polynucleotides selected from polynucleotides comprising or consistingof SEQ ID NOs: 1-57 or 94-148 or primers or probes specific for at least4 of SEQ ID NOs: 1-57 or 94-148. In another embodiment, the arraycomprises at least 5 polynucleotides selected from the polynucleotidescomprising or consisting of SEQ ID NOs: 1-57 or 94-148 or primers orprobes specific for at least 5 of SEQ ID NOs: 1-57 or 94-148. In anotherembodiment, the array comprises at least 10 polynucleotides selectedfrom the polynucleotides comprising or consisting of SEQ ID NOs: 1-57 or94-148 or primers or probes specific for at least 10 of SEQ ID NOs: 1-57or 94-148. In some embodiments, the array comprises the 24polynucleotides listed in Table 4 and/or primers and/or probes specificfor the 24 polynucleotides listed in Table 4. The array generallyincludes many copies of the selected polynucleotides to facilitatedetection. In some embodiments, the array comprises a million or morecopies of each of the selected polynucleotides.

In a particular embodiment, the array comprises or consists of SEQ IDNOs: listed in Table 4 or primers or probes specific for the SEQ ID NOs:listed in Table 4. Also encompassed herein is an array comprising orconsisting of subsets of SEQ ID NOs: listed in Table 4 or primers orprobes specific for these SEQ ID NOs: as set forth herein.

Probes for detecting polynucleotides described herein can be designedand prepared using conventional methods. Software for modeling anddesigning probes, including determining hybridization and annealingconditions, for detecting a specific polynucleotide sequence arepublically available, and include for example LightCycler® Probe DesignSoftware (Roche Applied Science), Primer3 (Simgene), and FastPCR(PrimerDigital). See also techniques described by Illumina (IlluminaInc., 5200 Illumina Way, San Diego, Calif. 92122, USA).

The array can include positive indicator for sepsis sequences and/orprobes for detecting same and negative and/or positive control sequencesand/or probes.

Polynucleotides described herein can be amplified and/or detected viaPCR, including but not limited to real-time PCR, multiplex PCR, nestedPCR, solid phase PCR, miniprimer PCR, and the like. Primers and probesfor amplifying and/or detecting polynucleotides described herein can bedesigned and prepared using conventional methods. Software for modelingand designing primers and probes, including determining hybridization,melting, annealing, and/or extensions conditions, for amplifying and/ordetecting a specific polynucleotide sequence are publicly available, andinclude for example LightCycler® Probe Design Software (Roche AppliedScience), Primer3 (Simgene), and FastPCR (PrimerDigital). See alsotechniques described by Illumina (Illumina Inc., 5200 Illumina Way, SanDiego, Calif. 92122, USA). PCR conditions generally include the presenceof four different nucleotide bases (adenosine, cytidine, guanosine,thymidine/uridine) and at least one polymerization-inducing agent suchas a reverse transcriptase or a DNA polymerase. The primers aregenerally present in a suitable buffer, which may include constituents,which are co-factors or affect conditions such as pH and the like atvarious suitable temperatures. The primers are preferably single-strandnucleotide sequences, such that amplification efficiency of the desiredpolynucleotide is optimized. Double-stranded nucleotide sequences canalso be utilized. The primers are typically at least about 15nucleotides. In some embodiments, the primers can have a length of fromabout 15 to about 30, about 15 to about 50, about 15 to about 75, about15 to about 100, or about 15 to about 500 nucleotides.

In some embodiments, primer sets are designed to amplify one or more ofthe sepsis-specific polynucleotides comprising or consisting of SEQ IDNOs: 1-57 or 94-148 and then the PCR products of the primer sets arescreened for sepsis-specific sequences on an array as described herein.

Diagnostic kits comprising one or more primer pairs, and optionalprobes, for amplifying and detecting one or more polynucleotidesdescribed herein are also provided. The kit can optionally includenucleotide bases (adenosine, cytidine, guanosine, thymidine/uridine) andat least one polymerization-inducing agent such as a reversetranscriptase or a DNA polymerase. The kit can optionally include asuitable primer buffer, which may include constituents which areco-factors or affect conditions such as pH and the like at varioussuitable temperatures. The kit can optionally include an array asdescribed herein.

The primers provided in the diagnostic kit are generally provided inpairs (forward primer and reverse primer) for amplifying/detecting aspecific polynucleotide sequence. These primers can be used to amplifyand detect CNAs in blood serum from humans, or any other appropriatebiological sample from humans that may contain CNAs. Alternatively, CNAscan be extracted from the sample and then amplified by PCR using adiagnostic kit of the disclosure. The CNA expression pattern ofsepsis-specific sequences detected by the diagnostic kit can be used todetermine if a human has early stage sepsis even at a stage wherein noclinical symptoms of sepsis are apparent

In addition to polynucleotide sequence specific primer pairs, labeledprobes specific for each of the CNAs may be included in kits describedherein. Labeled probes and use thereof in the context of, for example,multiplex qPCR provides for enhanced specificity of detection.

In some embodiments, the kit comprises primers for amplifying at least 2polynucleotides selected from the polynucleotides comprising orconsisting of SEQ ID NOs: 1-57 or 94-148, and optionally one or moreprobes for detecting the amplified product. In some embodiments, the kitcomprises primers for amplifying at least 4 polynucleotides selectedfrom the polynucleotides comprising or consisting of SEQ ID NOs: 1-57 or94-148, and optionally one or more probes for detecting the amplifiedproduct. In another embodiment, the kit comprises primers for amplifyingat least 5 polynucleotides selected from the polynucleotides comprisingor consisting of SEQ ID NOs: 1-57 or 94-148, and optionally one or moreprobes for detecting the amplified product. In another embodiment, thekit comprises primers for amplifying at least 10 polynucleotidesselected from the polynucleotides comprising or consisting of SEQ IDNOs: 1-57 or 94-148, and optionally one or more probes for detecting theamplified product. In some embodiments, the kit comprises primers foramplifying the 24 polynucleotides listed in Table 4, and optionally oneor more probes for detecting the amplified product.

In a particular embodiment, the kit comprises or consists of SEQ ID NOs:listed in Table 4 and/or primers and/or probes specific for the SEQ IDNOs: listed in Table 4. Also encompassed herein are kits comprising orconsisting of subsets of SEQ ID NOs: listed in Table 4 and/or primersand/or probes specific for these SEQ ID NOs: as set forth herein.

The present invention is described in the following Examples, which areset forth to aid in an understanding of the invention, and should not beconstrued to limit in any way the scope of the invention as defined inthe claims which follow thereafter.

EXAMPLES Example 1—Identification of Sepsis-Associated Polynucleotidesin CNAs

Serum from healthy individuals (controls) and diseased patients (e.g.,sepsis patients who were symptomatic or sepsis patients with early stagedisease, but were asymptomatic) was harvested and total DNA wasextracted from the serum samples. DNA was amplified and purified fromthe serum samples as described herein above. High-throughput paired-endDNA sequencing was performed and the resulting sequence reads weremapped to the human genome. Bioinformatics was used as described hereinto identify DNA motifs that were present at distinctively high or lowread count numbers in diseased humans, when compared to controls,corresponding to motifs that are over- and under-represented in sepsispatients. See, for example, Tables 1 and 3, which present motifs(Sequences) that are under-represented in sepsis patients (Table 1) orover-represented in sepsis patients (Table 3). The motifs identifiedreflect the host response to sepsis (i.e., the response of the humanbody to sepsis). The DNA motifs thus identified were used as targets forthe development of a real-time Polymerase-Chain Reaction (RT-PCR) assay.In addition to the motifs, which are present at distinctively differentlevels in healthy controls and sepsis patients, motifs that were presentat a highly similar level in healthy and diseased humans were alsoidentified to allow for the normalization of the results. See, forexample, Sequences presented in Table 2. The RT-PCR-evaluated motifs,which are used to discriminate between healthy controls and sepsispatients in a statistically significant manner, when evaluated alone orin combination with other motifs, are the subject basis for assaysdescribed herein.

All publications mentioned herein are hereby incorporated by referencein their entireties. While the foregoing invention has been described insome detail for purposes of clarity and understanding, it will beappreciated by one skilled in the art from a reading of the disclosurethat various changes in form and detail can be made without departingfrom the true scope of the invention in the appended claims.

Specific examples of methods and kits have been described herein forpurposes of illustration. These are only examples. The technologyprovided herein can be applied to systems other than the example systemsdescribed above. Many alterations, modifications, additions, omissions,and permutations are possible within the practice of this invention.This invention includes variations on described embodiments that wouldbe apparent to the skilled addressee, including variations obtained by:replacing features, elements and/or acts with equivalent features,elements and/or acts; mixing and matching of features, elements and/oracts from different embodiments; combining features, elements and/oracts from embodiments as described herein with features, elements and/oracts of other technology; and/or omitting combining features, elementsand/or acts from described embodiments.

The embodiments of the invention described above are intended to beexemplary only. Those skilled in this art will understand that variousmodifications of detail may be made to these embodiments, all of whichcome within the scope of the invention.

1. A method comprising administering to a human identified as havingsepsis a therapeutically effective amount of at least one agent used totreat sepsis, wherein the human is identified as having sepsis byanalyzing a biological sample isolated from the human forover-representation or under-representation of at least onepolynucleotide relative to an internal reference region, wherein the atleast one polynucleotide comprises any one of SEQ ID NOs: 1-57 or 94-148and wherein the over-representation or under-representation of the atleast one polynucleotide in the biological sample is indicative ofsepsis, thereby identifying the human as having sepsis.
 2. The method ofclaim 1, wherein the at least one polynucleotide comprising any one ofSEQ ID NOs: 1-57 or 94-148 over-represented or under-representedrelative to the internal reference region is at least two, at leastthree, at least four, at least five, at least six, at least seven, atleast eight, at least nine, at least ten, at least eleven, at leasttwelve, at least thirteen, at least fourteen, at least fifteen, at leastsixteen, at least seventeen, at least eighteen, at least nineteen, atleast twenty, at least twenty-one, at least twenty-two, at leasttwenty-three, or at least twenty-four of the polynucleotides comprisingany one of SEQ ID NOs: 1-57 or 94-148.
 3. The method of claim 1, whereinthe biological sample is blood, a product derived from blood, or afraction derived from blood.
 4. The method of claim 1, wherein theinternal reference region comprises at least one polynucleotidecomprising at least one of SEQ ID NOs: 59, 61, or
 68. 5. The method ofclaim 1, wherein detecting the over-representation orunder-representation of the at least one polynucleotide relative to aninternal reference region comprises at least one of a polymerase chainreaction (PCR)-based detection method, a hybridization-based method,enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA),solid-phase enzyme immunoassay (EIA), mass spectrometry, or microarrayanalysis.
 6. The method of claim 5, wherein the PCR-based detectionmethod is performed using at least one primer pair, wherein each primerpair of the at least one primer pair is specific for any one of SEQ IDNOs: 1-57 or 94-148, and a primer pair specific for at least one of SEQID NOs: 59, 61, or
 68. 7. The method of claim 6, wherein the primer pairspecific for any one of SEQ ID NOs: 1-57 or 94-148 and the primer pairspecific for at least one of SEQ ID NOs: 59, 61, or 68 is any one of theprimer pairs presented in Tables 1-3.
 8. The method of claim 7, furthercomprising sequencing amplification products corresponding to any one ofSEQ ID NOs: 1-57 or 94-148 at least one of SEQ ID NOs: 59, 61, or 68generated by the PCR-based detection method.
 9. The method of claim 1,wherein the at least one polynucleotides comprise circulating nucleicacids.
 10. The method of claim 1, wherein the at least one agent used totreat sepsis comprises at least one of an antibiotic, anti-fungal agent,anti-viral agent, anti-parasitic agent, or fluids suitable forintravenous administration.
 11. A primer comprising a manmade nucleotidesequence that binds specifically to a polynucleotide comprising any oneof the SEQ ID NOs: listed in Table 4 and at least one manmade tagconjugated thereto, wherein the manmade nucleotide sequence is any oneof the polynucleotide primer sequences listed in Tables 1-3 or a variantthereof.
 12. The primer of claim 11, wherein the variant of any one ofthe polynucleotide primer sequences listed in Tables 1-3 is at least90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least95%, at least 96%, at least 97%, at least 98%, or at least 99% identicalto any one of the polynucleotide sequences listed in Table
 4. 13. Theprimer of claim 11, wherein the manmade tag is a detectable marker. 14.The primer of claim 11, the primer consisting essentially of orconsisting of a manmade nucleotide sequence that binds specifically to apolynucleotide of any one of the SEQ ID NOs: listed in Table 4 and atleast one manmade tag conjugated thereto, wherein the manmade nucleotidesequence is any one of the polynucleotide primer sequences listed inTables 1-3 or a variant thereof and at least one manmade tag conjugatedthereto, wherein the manmade nucleotide sequence is any one of thepolynucleotide primer sequences listed in Tables 1-3 or a variantthereof.
 15. The polynucleotide primer sequence of claim 14, wherein thevariant of any one of the polynucleotide primer sequences listed inTables 1-3 is at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, or at least 95%, at least 96%, at least 97%, at least 98%, orat least 99% identical to any one of the polynucleotide primer sequenceslisted in Tables 1-3.
 16. The primer of claim 14, wherein the manmadetag is a detectable marker.
 17. A kit for detecting sepsis in a humancomprising at least one primer pair of claim 11 and instructions for usethereof.
 18. The kit of claim 17, wherein the at least one primer paircomprises four primer pairs and wherein each primer pair of the fourprimer pairs specifically amplifies a different polynucleotidecomprising any one of the SEQ ID NOs: listed in Table 4.